TW202313660A

TW202313660A - Variant staphylococcus aureus luka and lukb polypeptides and vaccine compositions

Info

Publication number: TW202313660A
Application number: TW110136365A
Authority: TW
Inventors: 某瑞布萊恩; 康斯坦丁諾夫謝爾蓋; 格森杰倫; 羅金泉; 索馬尼桑迪普; Ｔ巴克利彼得; Ｊ托雷斯維克特
Original assignee: 美商楊森製藥公司; 紐約大學
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2023-04-01

Abstract

This invention relates to Staphylococcus aureus leukocidin A (LukA) and leukocidin B (LukB) variant polypeptides and polynucleotides encoding the LukA, LukB and LukAB variant polypeptides. This invention further relates to vaccine compositions containing these LukA and LukB variants, as well as methods for generating immune responses against Staphylococcus aureus in subjects.

Description

變異的金黃色葡萄球菌LukA和LukB多肽和疫苗組合物Variant Staphylococcus aureus LukA and LukB polypeptides and vaccine compositions

本發明涉及變異的金黃色葡萄球菌殺白細胞素A (LukA)和殺白細胞素B (LukB)蛋白和多肽、包含這些LukA和LukB變體的疫苗組合物，以及所述疫苗組合物在受試者中誘導免疫應答以治療和/或預防金黃色葡萄球菌感染的用途。The present invention relates to variant Staphylococcus aureus leukocidin A (LukA) and leukocidin B (LukB) proteins and polypeptides, vaccine compositions comprising these LukA and LukB variants, and the vaccine composition in subjects Induction of an immune response for the treatment and/or prevention of Staphylococcus aureus infection.

金黃色葡萄球菌引起廣泛的侵襲性疾病，包括敗血症、感染性心內膜炎和中毒性休克，以及不太嚴重的皮膚和軟組織感染(Tong等人，“ Staphylococcus aureusInfections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management,” Clin. Microbiol. Rev. 28(3):603-661 (2015))。目前，沒有批准用於對抗金黃色葡萄球菌的疫苗，治療選擇受到新出現的抗生素耐藥性的進一步限制(Sause等人，“Antibody-Based Biologics and Their Promise to Combat Staphylococcus aureusInfections,” Trends Pharmacol. Sci. 37(3):231-241 (2016))。金黃色葡萄球菌引起多種臨床綜合征的能力通常與基因組含量的主要變化有關(Copin等人，“After the Deluge: Mining Staphylococcus aureusGenomic Data for Clinical Associations and Host-Pathogen Interactions,” Curr. Opin. Microbiol. 41:43-50 (2018) 和Recker等人, “Clonal Differences in Staphylococcus aureusBacteraemia-Associated Mortality,” Nat. Microbiol. 2(10):1381-1388 (2017))。值得注意的是，大約40%的基因組不是所有金黃色葡萄球菌分離株所共有的(Bosi等人，“Comparative Genome-Scale Modelling of Staphylococcus aureusStrains Identifies Strain-Specific Metabolic Capabilities Linked to Pathogenicity,” Proc. Natl. Acad. Sci. USA 113(26):E3801-3809 (2016))，因而使疫苗和生物制品生產的保守靶標的鑒定更加複雜。 Staphylococcus aureus causes a wide range of invasive diseases, including sepsis, infective endocarditis, and toxic shock, as well as less serious skin and soft tissue infections (Tong et al., " Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations , and Management,” Clin. Microbiol. Rev. 28(3):603-661 (2015)). Currently, there are no approved vaccines against S. aureus, and treatment options are further limited by emerging antibiotic resistance (Sause et al., “Antibody-Based Biologics and Their Promise to Combat Staphylococcus aureus Infections,” Trends Pharmacol. Sci. 37(3):231-241 (2016)). The ability of S. aureus to cause multiple clinical syndromes is often associated with major changes in genome content (Copin et al., "After the Deluge: Mining Staphylococcus aureus Genomic Data for Clinical Associations and Host-Pathogen Interactions," Curr. Opin. Microbiol. 41:43-50 (2018) and Recker et al., “Clonal Differences in Staphylococcus aureus Bacteraemia-Associated Mortality,” Nat. Microbiol. 2(10):1381-1388 (2017)). Notably, approximately 40% of the genome is not shared by all S. aureus isolates (Bosi et al., “Comparative Genome-Scale Modeling of Staphylococcus aureus Strains Identifies Strain-Specific Metabolic Capabilities Linked to Pathogenicity,” Proc. Natl . Acad. Sci. USA 113(26):E3801-3809 (2016)), thus complicating the identification of conserved targets for vaccine and biologics production.

本發明旨在克服本領域中的這些和其他限制。The present invention seeks to overcome these and other limitations in the art.

本發明的第一方面涉及變異的金黃色葡萄球菌殺白細胞素A蛋白或其多肽。在一個方面，所述LukA變體是SEQ ID NO:25的LukA蛋白或多肽的變體，包括對應於SEQ ID NO:25的氨基酸殘基Lys83、Ser141、Val113、Val193的一個或多個氨基酸殘基上的氨基酸取代。A first aspect of the present invention relates to a variant S. aureus leukocidin A protein or a polypeptide thereof. In one aspect, the LukA variant is a variant of the LukA protein or polypeptide of SEQ ID NO:25 comprising one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113, Val193 of SEQ ID NO:25 Amino acid substitutions on the base.

本發明的其他方面涉及具有一個或多個附加氨基酸取代、缺失和/或添加至上述氨基酸的LukA變體。本發明還涉及編碼變異LukA蛋白或多肽的核酸分子，以及包含前述核酸分子的表達載體。Other aspects of the invention relate to LukA variants having one or more additional amino acid substitutions, deletions and/or additions to the aforementioned amino acids. The present invention also relates to a nucleic acid molecule encoding a variant LukA protein or polypeptide, and an expression vector comprising the aforementioned nucleic acid molecule.

本發明的另一方面涉及變異的金黃色葡萄球菌殺白細胞素B蛋白或其多肽。在一個方面，LukB變體是SEQ ID NO:39的LukB蛋白或多肽的變體，包含對應於SEQ ID NO:39的氨基酸殘基Val53上的氨基酸取代。Another aspect of the invention relates to variant S. aureus leukocidin B proteins or polypeptides thereof. In one aspect, the LukB variant is a variant of the LukB protein or polypeptide of SEQ ID NO:39 comprising an amino acid substitution corresponding to amino acid residue Val53 of SEQ ID NO:39.

在一個方面，LukB變體是SEQ ID NO:39的LukB蛋白或多肽的變體，包含對應於氨基酸殘基Glu45、Glu109、Thr121和Arg154的氨基酸殘基上的氨基酸取代。In one aspect, the LukB variant is a variant of the LukB protein or polypeptide of SEQ ID NO: 39 comprising amino acid substitutions at amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121 and Arg154.

本發明的其他方面涉及具有一個或多個附加氨基酸取代、缺失和/或添加至上述氨基酸的LukB變體。本發明還涉及編碼變異LukB蛋白或多肽的核酸分子，以及包含前述核酸分子的表達載體。Other aspects of the invention relate to LukB variants having one or more additional amino acid substitutions, deletions and/or additions to the aforementioned amino acids. The present invention also relates to a nucleic acid molecule encoding a variant LukB protein or polypeptide, and an expression vector comprising the aforementioned nucleic acid molecule.

本發明的另一方面涉及一種表達載體，所述表達載體包含編碼本文所述LukA變異多肽的核酸分子，所述核酸分子可操作地與編碼本文所述LukB多肽或LukB變異多肽的核酸分子偶聯。Another aspect of the present invention relates to an expression vector, the expression vector comprises a nucleic acid molecule encoding a LukA variant polypeptide described herein, and the nucleic acid molecule is operably coupled to a nucleic acid molecule encoding a LukB polypeptide or a LukB variant polypeptide described herein .

本發明的另一方面涉及包含本文所述的任何一種或多種表達載體的宿主細胞。Another aspect of the invention pertains to host cells comprising any one or more expression vectors described herein.

本發明的另一個方面涉及包含本文所述LukA變異多肽的金黃色葡萄球菌疫苗組合物。Another aspect of the invention pertains to S. aureus vaccine compositions comprising the LukA variant polypeptides described herein.

本發明的另一方面涉及包含本文所述的LukB變異多肽的金黃色葡萄球菌疫苗組合物。Another aspect of the invention relates to S. aureus vaccine compositions comprising the LukB variant polypeptides described herein.

本發明的另一個方面涉及金黃色葡萄球菌疫苗組合物，包含本文所述的LukA變異多肽和本文所述的LukB變異多肽。Another aspect of the present invention relates to a Staphylococcus aureus vaccine composition comprising the LukA variant polypeptide described herein and the LukB variant polypeptide described herein.

本發明的另一方面涉及在受試者中產生針對金黃色葡萄球菌的免疫應答的方法。所述方法包括在所述受試者中可有效產生針對金黃色葡萄球菌的所述免疫應答的條件下，向所述受試者施用本文所述的疫苗組合物。Another aspect of the invention relates to methods of generating an immune response against S. aureus in a subject. The method comprises administering to the subject a vaccine composition described herein under conditions effective to generate the immune response against S. aureus in the subject.

金黃色葡萄球菌(S. aureus)是導致大量醫院和社區獲得性感染的原因。為了逃避免疫系統的清除，金黃色葡萄球菌采用多種方法，包括分泌被稱為殺白細胞素的雙組分成孔毒素。每個殺白細胞素由兩個約300個氨基酸長的多肽組成，分為宿主細胞靶向(S型，慢，基於其色譜洗脫曲線)和聚合作用(F型，快)的亞單位。在人類金黃色葡萄球菌分離株中已經記載了多達五種殺白細胞素:Panton-Valentine殺白細胞素(PVL，或LukSF-PV)、γ-溶血素(HlgAB和HlgCB)、殺白細胞素ED (LukED)和殺白細胞素AB(LukB，也稱為LukGH)。殺白細胞素與特定的細胞表面蛋白受體結合，並組裝成低聚孔，最終由於快速滲透失調而導致細胞溶解(Spaan等人，“Leukocidins: Staphylococcal Bi-Component Pore-Forming Toxins find their Receptors” Nat. Rev. Microbiol. 15(7):435-447(2017))。Staphylococcus aureus (S. aureus) is responsible for a large number of hospital and community-acquired infections. To evade clearance by the immune system, S. aureus employs a variety of methods, including secreting a two-component pore-forming toxin called leukocidin. Each leukocidin consists of two approximately 300 amino acid long polypeptides divided into subunits for host cell targeting (S-type, slow, based on their chromatographic elution profile) and aggregation (F-type, fast). Up to five leukocidins have been documented in human S. aureus isolates: Panton-Valentine leukocidin (PVL, or LukSF-PV), γ-hemolysin (HlgAB and HlgCB), leukocidin ED ( LukED) and Leukocidin AB (LukB, also known as LukGH). Leukocidins bind to specific cell surface protein receptors and assemble into oligomeric pores that eventually lead to cell lysis due to rapid osmotic dysregulation (Spaan et al., "Leukocidins: Staphylococcal Bi-Component Pore-Forming Toxins find their Receptors" Nat . Rev. Microbiol. 15(7):435-447(2017)).

最近發現的殺白細胞素，LukAB (DuMont等，“Characterization of a New Cytotoxin that Contributes to Staphylococcus aureus Pathogenesis” Mol. Microbiol. 79(3):814-825 (2011))，具有幾個區別於其他雙組分殺白細胞素的獨特特征。LukA (S型)和LukB (F型)亞單位作為預組裝的二聚體存在於溶液中，而不是單獨的單體(DuMont等人，“Identification of a Crucial Residue Required for Staphylococcus aureus LukAB Cytotoxicity and Receptor Recognition” Infection and Immunity 82(3):1268-1276 (2014))。而且，LukAB靶向宿主細胞表面的CD11b/CD18整合素(DuMont等，“Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1” Proc Natl Acad Sci USA. 110(26):10794-9 (2013))，不同於PVL、LukED、HlgAB和HlgCB，它們與特定的七種跨膜趨化因子受體相互作用(Spaan等人，“Leukocidins: Staphylococcal Bi-Component Pore-Forming Toxins find their Receptors” Nat. Rev. Microbiol. 15(7):435-447(2017))。最後，LukAB是在使用了原代人白細胞的體外感染模型中負責金黃色葡萄球菌介導的細胞裂解的主要毒素(DuMont等人，“Characterization of a New Cytotoxin that Contributes to Staphylococcus aureus Pathogenesis” Mol. Microbiol. 79(3):814-825 (2011))。這些獨特的特征可能是由LukAB與其他殺白細胞素的差異來解釋的。通常的，在每組中，S型和F型成分分別具有71-82%和65-81%的同一性，而LukA和LukB與其他殺白細胞素只有30%和39%的同一性。重要的是，殺白細胞素AB (LukAB)存在於迄今為止記載的所有人類感染分離株中，並且在最遠的葡萄球菌譜系之間表現出高達20%的氨基酸差異。The recently discovered leukocidin, LukAB (DuMont et al., "Characterization of a New Cytotoxin that Contributes to Staphylococcus aureus Pathogenesis" Mol. Microbiol. 79(3):814-825 (2011)), has several differences from other bigroups The unique characteristics of leukocidin. LukA (S-type) and LukB (F-type) subunits exist in solution as preassembled dimers rather than individual monomers (DuMont et al., "Identification of a Crucial Residue Required for Staphylococcus aureus LukAB Cytotoxicity and Receptor Recognition” Infection and Immunity 82(3):1268-1276 (2014)). Furthermore, LukAB targets the CD11b/CD18 integrin on the host cell surface (DuMont et al., "Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1" Proc Natl Acad Sci USA. 110(26):10794 -9 (2013)), which, unlike PVL, LukED, HlgAB, and HlgCB, interact with specific seven transmembrane chemokine receptors (Spaan et al., "Leukococcal Bi-Component Pore-Forming Toxins find their Receptors” Nat. Rev. Microbiol. 15(7):435-447(2017)). Finally, LukAB is the major toxin responsible for S. aureus-mediated cell lysis in an in vitro infection model using primary human leukocytes (DuMont et al., "Characterization of a New Cytotoxin that Contributes to Staphylococcus aureus Pathogenesis" Mol. Microbiol 79(3):814-825 (2011). These unique features may be explained by differences of LukAB from other leukocidins. Typically, within each group, the S- and F-type components share 71-82% and 65-81% identities, respectively, while LukA and LukB share only 30% and 39% identities with other leukocidins. Importantly, leukocidin AB (LukAB) is present in all human infection isolates documented to date and exhibits up to 20% amino acid variation between the most distant staphylococcal lineages.

鑒於LukAB毒素在金黃色葡萄球菌發病機理和感染中的重要性，本文公開了新的LukA和LukB變異蛋白和多肽。本文公開的LukA和LukB變異蛋白和多肽保留了它們相互之間二聚化的能力，因為它們可以維持天然毒素結構並向免疫系統呈現以產生強大的免疫反應使它們成為理想的疫苗候選物，但缺點是缺乏細胞毒性活性。鑒於每年有大量的人感染金黃色葡萄球菌，這些感染中很大一部分很可能對傳統的抗生素治療無效。本文記載的治療，更重要的是預防此類感染的創新方法包括抑制金黃色葡萄球菌毒力因子，如LukAB，其負責殺死多形核白細胞(PMNs)，多形核白細胞(PMNs)是參與防禦金黃色葡萄球菌感染的最關鍵的先天免疫細胞。In view of the importance of the LukAB toxin in the pathogenesis and infection of Staphylococcus aureus, novel LukA and LukB variant proteins and polypeptides are disclosed herein. The LukA and LukB variant proteins and polypeptides disclosed herein retain their ability to dimerize with each other because they can maintain the native toxin structure and present it to the immune system to generate a robust immune response making them ideal vaccine candidates, but The disadvantage is the lack of cytotoxic activity. Given the large number of people infected with S. aureus each year, it is likely that a large proportion of these infections will not respond to traditional antibiotic treatment. The innovative approach documented here to treat, and more importantly prevent, such infections involves inhibiting S. aureus virulence factors, such as LukAB, which are responsible for killing polymorphonuclear leukocytes (PMNs), which are involved in The most critical innate immune cell for defense against Staphylococcus aureus infection.

本發明涉及變異的金黃色葡萄球菌殺白細胞素蛋白和多肽以及包含這些變異蛋白和多肽的組合物。本發明還涉及一種預防受試者金黃色葡萄球菌感染的方法。The present invention relates to variant S. aureus leukocidin proteins and polypeptides and compositions comprising these variant proteins and polypeptides. The invention also relates to a method of preventing S. aureus infection in a subject.

定義definition

在描述本發明的組合物和方法之前，應當理解，本發明不限於所描述的特定組合物或方法，因為它們可以變化。還應當理解，說明書中使用的術語僅用於描述特定方案或實施例的目的，並不旨在限制本文實施例的範圍，本發明實施例的範圍將僅由所附申請專利範圍來限制。除非另有定義，本文使用的所有技術和科學術語與本領域普通技術人員通常理解的含義相同。盡管與本文描述的方法和材料相似或等同的任何方法和材料可用於本文實施例的實踐或測試，但是現在描述優選的方法、裝置和材料。本文中提到的所有出版物均通過引用整體並入本文。本文中的任何內容均不應被解釋為承認本文中的實施例無權憑借現有發明先於此類公開。Before the compositions and methods of the present invention are described, it is to be understood that this invention is not limited to particular compositions or methods described, as these may vary. It should also be understood that the terminology used in the specification is only used for the purpose of describing specific solutions or embodiments, and is not intended to limit the scope of the embodiments herein. The scope of the embodiments of the present invention will only be limited by the appended patent scope. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments herein, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the examples herein are not entitled to antedate such disclosure by virtue of prior invention.

必須注意的是，如本文和所附申請專利範圍中所使用的，單數形式“一個”、“一種”和“該”包括複數指代形式，除非上下文另有明確規定。It must be noted that, as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

除非另有說明，否則任何數值，如本文所述的濃度或濃度範圍，應理解為在所有情況下都被術語“約”修飾。因此，數值通常包括所述值的10%浮動範圍。例如，1mg/ml的濃度包括0.9mg/ml~1.1mg/ml。同樣，1%~10%(w/v)的濃度範圍包括0.9%(w/v)~11%(w/v)。如本文所使用的，數值範圍的使用明確地包括所有可能的子範圍，該範圍內的所有單個數值，包括數值在該範圍內的整數和分數，除非上下文明確地另外指出。Unless otherwise indicated, any numerical value, such as a concentration or concentration range stated herein, is to be understood as being modified in all instances by the term "about". Accordingly, numerical values generally include a 10% variation of the stated value. For example, a concentration of 1 mg/ml includes 0.9 mg/ml to 1.1 mg/ml. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges and all individual values within that range, including integers and fractions of values within that range, unless the context clearly dictates otherwise.

除非另有說明，一系列要素之前的術語“至少”應理解為指該系列中的每個要素。本領域技術人員將認識到或能夠僅使用常規實驗來確定這裏描述的本發明的具體實施例的許多等同物。這些等同物旨在包含在本發明中。Unless stated otherwise, the term "at least" preceding a series of elements should be understood as referring to each element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by this invention.

如本文所使用的，術語“包含（comprises）”、“含有（comprising）”、“包括（includes）”、“包括（including）”、“具有”、“蘊含”、“由...構成”、“由...組成”或其任何其他不同表述，將被理解為包含所述整數或整數組，但不排除任何其他整數或整數組，並且旨在是非排他性的或開放式的。例如，包括一系列要素的組合物、混合物、工藝、方法、制品或設備不一定僅限於這些要素，而是可以包括未明確列出的或這種組合物、混合物、工藝、方法、制品或設備固有的其他要素。此外，除非有相反的明確說明，“或”指的是包含性的或，而不是排他性的或。例如，條件A或B由以下任一項滿足:A為真(或存在)，B為假(或不存在)，A為假(或不存在)，B為真(或存在)，A和B都為真(或存在)。如本文所使用的，多個引用的要素之間的連接術語“和/或”被理解為包括單獨的和組合的選項。例如，當兩個要素通過“和/或”連接在一起時，第一個選項指的是第一個要素的適用性，而沒有第二個要素。第二種選擇是指沒有第一種要素的第二種要素的適用性。第三種選擇是指第一和第二要素一起的適用性。這些選項中的任何一個被理解為落入該含義內，因此滿足這裏使用的術語“和/或”的要求。一個以上選項的同時適用性也被理解為落入該含義內，因此滿足術語“和/或”的要求。As used herein, the terms "comprises", "comprising", "includes", "including", "has", "implies", "consists of" , "consisting of" or any other variation thereof, will be understood to include said integer or group of integers, but not exclude any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that includes a list of elements is not necessarily limited to those elements, but may include items not expressly listed or such composition, mixture, process, method, article, or apparatus Inherent other elements. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, the condition A or B is satisfied by any of the following: A is true (or exists), B is false (or does not exist), A is false (or does not exist), B is true (or exists), A and B are both true (or exist). As used herein, the linking term "and/or" between multiple referenced elements is understood to include both individual and combined options. For example, when two elements are joined by "and/or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within that meaning, thus satisfying the requirements of the term "and/or" as used herein. The simultaneous applicability of more than one option is also understood to fall within this meaning, thus satisfying the requirements of the term "and/or".

如本文所用，術語“由...組成”或其不同表述諸如“由...構成”或“其組成為”，如在整個說明書和申請專利範圍中所使用的，表示包括任何引用的整數或整數組，但是沒有額外的整數或整數組可以添加到特定的方法、結構或組成中。As used herein, the term "consisting of" or its variations such as "consisting of" or "consisting of", as used throughout the specification and claims, means to include any cited integer or groups of integers, but no additional integers or groups of integers may be added to a particular method, structure, or composition.

如本文所使用的，術語“基本上由...組成（consists essentially of）”或不同表述，例如貫穿說明書和申請專利範圍所使用的“基本上由...構成（consist essentially of）”或“基本上由...組成（consisting essentially of）”，表示包括任何列舉的整數或整數組，以及任選包括任何列舉的整數或整數組，其不會實質上改變特定方法、結構或組成的基本或新穎性。As used herein, the term "consists essentially of" or different expressions, such as "consist essentially of" or "consist essentially of" used throughout the specification and claims "Consisting essentially of" means including any recited integer or group of integers, and optionally including any recited integer or group of integers that does not materially alter the particular method, structure, or composition Basic or novel.

如本文所用，“受試者”是指任何動物，優選哺乳動物，最優選人。本文使用的術語“哺乳動物”包括任何哺乳動物。哺乳動物的示例包括但不限於牛、馬、羊、豬、貓、狗、小鼠、大鼠、兔子、豚鼠、猴子、人類等，更優選人類。As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

還應該理解的是，本文使用的術語“大約”、“近似”、“一般”、“基本上/實質上”和類似術語，當指優選發明的部件的尺寸或特性時，表示所述尺寸/特性不是嚴格的範圍或參數，並且不排除功能上相同或相似的微小變化，如本領域普通技術人員所理解的。至少，包括數值參數的這種參考將包括使用本領域接受的數學和工業原理的變化(例如，舍入、測量或其他系統誤差、制造公差等)，不會改變最低有效數字。It should also be understood that as used herein, the terms "about", "approximately", "generally", "substantially/substantially" and similar terms, when referring to a dimension or characteristic of a preferred inventive component, mean that said dimension/ The characteristics are not strict ranges or parameters, and do not exclude functionally identical or similar minor variations, as understood by those of ordinary skill in the art. At a minimum, such references including numerical parameters will include changes using mathematical and industry principles accepted in the art (eg, rounding, errors of measurement or other systematics, manufacturing tolerances, etc.) that will not alter the least significant digit.

在兩種或多種核酸或多肽序列(例如，葡萄球菌LukA和LukB多肽以及編碼它們的多核苷酸)的上下文中，術語“完全相同”或“同一性”百分比是指兩種或多種序列或子序列，當使用以下序列比較算法之一或通過目測進行比較和比對以獲得最大對應性時，它們是相同的或具有相同的特定百分比的氨基酸殘基或核苷酸。In the context of two or more nucleic acid or polypeptide sequences (e.g., staphylococcal LukA and LukB polypeptides and polynucleotides encoding them), the term "identical" or "identity" percentage refers to two or more sequences or subunits. Sequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms or by visual inspection.

對於序列比較，通常將一個序列作為參考序列，測試序列與之進行比較。當使用序列比較算法時，測試和參考序列被輸入到計算機中，必要時指定子序列坐標，並且指定序列算法程序參數。然後，序列比較算法根據指定的程序參數，計算測試序列相對於參考序列的序列同一性百分比。For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the designated program parameters.

用於比較的序列的最佳比對方法可以通過下述方法進行：例如通過Smith & Waterma的局部同源性算法(Adv. Appl. Math. 2:482 (1981))，通過Needleman & Wunsch的同源性比對算法( J. Mol. Biol. 48:443 (1970))，通過Pearson & Lipman的相似性搜索方法(Proc. Nat'l. Acad. Sci. USA 85:2444 (1988))，這些算法通過計算機化(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI)，或通過視覺檢查(一般參見，Current Protocols in Molecular Biology, F.M. Ausubel等人，eds., Current Protocols, (1995 Supplement))實現。Methods of optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterma (Adv. Appl. Math. 2:482 (1981)), by the same method of Needleman & Wunsch Genetic comparison algorithm ( J. Mol. Biol. 48:443 (1970)), through Pearson & Lipman's similarity search method (Proc. Nat'l. Acad. Sci. USA 85:2444 (1988)), these Algorithms were analyzed by computerization (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, (1995 Supplement)).

適用於確定序列同一性百分比和序列相似性的算法的例子是BLAST和BLAST 2.0算法，它們分別記載於Altschul等人((1990) J. Mol. Biol. 215: 403-410) 和Altschul等人((1997) Nucleic Acids Res. 25: 3389- 3402)的研究中。用於執行BLAST分析的軟件可通過國家生物技術信息中心公開獲得。Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. ((1990) J. Mol. Biol. 215: 403-410) and Altschul et al. ( (1997) Nucleic Acids Res. 25: 3389- 3402). Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information.

除了計算序列同一性百分比，BLAST算法還對兩個序列之間的相似性進行統計分析(例如，參見Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993))。BLAST算法提供的一種相似性測量是最小和概率(P(N))，它提供了兩個核苷酸或氨基酸序列之間偶然發生匹配的概率的指示。例如，如果測試核酸與參考核酸的比較中的最小和概率小於約0.1，更優選小於約0.01，最優選小於約0.001，則認為核酸類似於參考序列。In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)) . One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

如下所述，兩個核酸序列或多肽基本相同的進一步指示是由第一個核酸編碼的多肽與由第二個核酸編碼的多肽在免疫學上可進行交叉反應。因此，一種多肽通常與第二種多肽基本相同，例如，這兩種多肽僅在保守取代上有所不同。兩個核酸序列基本相同的另一個指示是這兩個分子在嚴格的條件下可相互雜交。A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, one polypeptide is typically substantially identical to a second polypeptide, eg, the two polypeptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

如本文所用，術語“多核苷酸”，同義地稱為“核酸分子”、“核苷酸”或“核酸”，是指任何多核糖核苷酸或多脫氧核糖核苷酸，其可以是未修飾的RNA或DNA或修飾的RNA或DNA。“多核苷酸”包括但不限於單鏈和雙鏈DNA、單鏈和雙鏈區域的混合物DNA、單鏈和雙鏈RNA、單鏈和雙鏈區域的混合物RNA、包含單鏈或更典型地雙鏈的DNA和RNA的雜交分子或單鏈和雙鏈區域的混合物。此外，“多核苷酸”是指包含RNA或DNA或RNA和DNA的三鏈區域。術語多核苷酸還包括含有一個或多個修飾鹼基的DNA或RNA，以及為了穩定性或其他原因而進行骨架修飾的DNA或RNA。“修飾的”鹼基包括，例如，三苯甲基化的鹼基和獨特的鹼基，如肌苷。可以對DNA和RNA進行多種修飾；因此，“多核苷酸”包括通常在自然界中發現的多核苷酸的化學、酶促或代謝修飾形式，以及病毒和細胞特有的DNA和RNA的化學形式。“多核苷酸”也包括相對較短的核酸鏈，通常稱為寡核苷酸。As used herein, the term "polynucleotide", synonymously referred to as "nucleic acid molecule", "nucleotide" or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which may be Modified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, mixtures of single- and double-stranded regions DNA, single- and double-stranded RNA, mixtures of single- and double-stranded regions A hybrid molecule of double-stranded DNA and RNA or a mixture of single- and double-stranded regions. Furthermore, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA backbone modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unique bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also includes relatively short strands of nucleic acid, commonly referred to as oligonucleotides.

如本文所用，術語“載體”是指例如任意數量的核酸，其中可以***所需序列，例如限制性和連接，用於在遺傳環境之間運輸或在宿主細胞中表達。核酸載體可以是DNA或RNA。載體包括但不限於質粒、噬菌體、噬菌粒、細菌基因組、病毒基因組、自擴增RNA、複制子。As used herein, the term "vector" refers to, for example, any number of nucleic acids into which desired sequences can be inserted, eg, restricted and ligated, for transport between genetic environments or for expression in a host cell. A nucleic acid vector can be DNA or RNA. Vectors include, but are not limited to, plasmids, phages, phagemids, bacterial genomes, viral genomes, self-amplifying RNA, replicons.

如本文所用，術語“宿主細胞”是指包含本發明核酸分子的細胞。“宿主細胞”可以是任何類型的細胞，例如原代細胞、培養的細胞或來自細胞系的細胞。在一個實施方案中，“宿主細胞”是用本發明的核酸分子轉染或轉導的細胞。在另一個實施方案中，“宿主細胞”是這種轉染或轉導細胞的後代或潛在後代。細胞的子代可能與親代細胞相同，也可能不同，例如，由於突變或環境影響(可能發生在子代中)或核酸分子整合到宿主細胞基因組中。As used herein, the term "host cell" refers to a cell comprising a nucleic acid molecule of the invention. A "host cell" can be any type of cell, such as a primary cell, a cultured cell, or a cell from a cell line. In one embodiment, a "host cell" is a cell transfected or transduced with a nucleic acid molecule of the invention. In another embodiment, a "host cell" is the progeny or potential progeny of such transfected or transduced cells. The progeny of a cell may or may not be identical to the parent cell, for example, due to mutations or environmental influences (which may occur in progeny) or the integration of nucleic acid molecules into the host cell genome.

本文所用，術語“表達”是指基因產物的生物合成。該術語包括將基因轉錄成RNA。該術語還包括將RNA翻譯成一種或多種多肽，還包括所有天然存在的轉錄後和翻譯後的修飾。表達的多肽可以在宿主細胞的細胞質內，進入細胞外環境，如細胞培養物的生長培養基，或固定在細胞膜上。As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes the transcription of a gene into RNA. The term also includes translation of RNA into one or more polypeptides, and also includes all naturally occurring post-transcriptional and post-translational modifications. The expressed polypeptide can be within the cytoplasm of the host cell, enter the extracellular environment, such as the growth medium of a cell culture, or be immobilized on the cell membrane.

如本文所用，術語“肽”、“多肽”或“蛋白質”可以指由氨基酸組成的分子，並且可以被本領域技術人員認為為蛋白質。術語“肽”、“多肽”和“蛋白質”在本文中可以互換使用，指任何長度的氨基酸聚合物。聚合物可以是直鏈或支鏈的，它可以包含修飾的氨基酸，並且它可以被非氨基酸中斷。該術語還包括天然的或通過幹預修飾的氨基酸聚合物；例如二硫鍵形成、糖基化、脂質化、乙醯化、磷酸化或任何其他操作或修飾，例如與標記成分結合。定義還包括例如含有一種或多種氨基酸類似物(包括例如非天然氨基酸等)的多肽，以及本領域已知的其他修飾。As used herein, the term "peptide", "polypeptide" or "protein" may refer to a molecule composed of amino acids and may be considered a protein by those skilled in the art. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. A polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that are natural or modified by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as incorporation of a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

本文所述的多肽序列是根據通常的慣例書寫的，其中肽的N-末端區域在左邊，C-末端區域在右邊。盡管氨基酸的異構形式是已知的，但是除非另另有明確說明，否則所代表的是氨基酸的左旋形式。Polypeptide sequences described herein are written according to the usual convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomeric forms of amino acids are known, unless expressly stated otherwise, the left-handed form of the amino acid is represented.

術語“分離的”可以指基本上不含細胞物質、細菌物質、病毒物質或其原始來源的培養基(當通過重組DNA技術產生時)、或化學前體或其他化學物質(當化學合成時)的核酸或多肽。此外，分離的多肽是指可以作為分離的多肽給予受試者的多肽；換句話說，如果多肽粘附在柱上或嵌入凝膠中，就不能簡單地認為它是“分離的”。此外，“分離的核酸片段”或“分離的肽”是並非作為片段天然存在和/或通常不處於功能狀態的核酸或蛋白質片段。The term "isolated" may refer to a culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized) that is substantially free of cellular material, bacterial material, viral material, or their original source Nucleic acid or polypeptide. Furthermore, an isolated polypeptide refers to a polypeptide that can be administered to a subject as an isolated polypeptide; in other words, a polypeptide cannot simply be considered "isolated" if it is adhered to a column or embedded in a gel. Furthermore, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not occur naturally as a fragment and/or is not normally in a functional state.

如本文所用，短語“免疫應答”或其等同的表述“免疫學應答”是指在受試者中針對本發明的蛋白質、肽、碳水化合物或多肽的體液(抗體介導的)、細胞(由抗原特異性T細胞或其分泌產物介導的)或體液和細胞應答的發展。這種應答可以是通過施用免疫原誘導的主動應答，也可以是通過施用抗體、含有抗體的物質或活化的T細胞誘導的被動應答。細胞免疫應答是通過與第一類或第二類MHC分子相關的多肽表位的呈遞而引發的，以活化抗原特異性CD4 (+)輔助性T細胞和/或CD8 (+)細胞毒性T細胞。這種應答還可能涉及單核細胞、巨噬細胞、自然殺傷細胞、嗜鹼性粒細胞、樹突狀細胞、星形膠質細胞、小膠質細胞、嗜酸性粒細胞或先天免疫的其他成分的活化。本文所用的“主動免疫”是指通過施用抗原而賦予受試者的任何免疫。As used herein, the phrase "immune response" or its equivalent expression "immunological response" refers to a humoral (antibody-mediated), cellular ( mediated by antigen-specific T cells or their secreted products) or the development of humoral and cellular responses. This response may be an active response induced by the administration of an immunogen, or a passive response induced by the administration of an antibody, antibody-containing substance or activated T cells. The cellular immune response is elicited by the presentation of polypeptide epitopes associated with class I or class II MHC molecules to activate antigen-specific CD4(+) helper T cells and/or CD8(+) cytotoxic T cells . This response may also involve the activation of monocytes, macrophages, natural killer cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or other components of innate immunity . "Active immunization" as used herein refers to any immunity imparted to a subject by administration of an antigen.

金黃色葡萄球菌殺白細胞素Staphylococcus aureus leukocidin AA 變體Variants

本發明的第一方面涉及變體，即非天然存在的金黃色葡萄球菌( S. aureus)殺白細胞素A (LukA)蛋白質或多肽。這些變異的LukA蛋白或多肽包含一個或多個氨基酸殘基的***、取代和/或缺失，使得LukAB雙組分毒素無細胞毒性，穩定LukAB異二聚體，提高解鏈溫度，和/或增加溶解度。如本文所述，這些變異的LukA蛋白和多肽是理想的疫苗抗原候選物，可以單獨給藥或與殺白細胞素B (LukB)野生型或變異蛋白或多肽聯合給藥。當與LukB蛋白或其多肽聯合給藥時，所得類毒素模擬金黃色葡萄球菌LukB毒素的結構，從而促進產生針對金黃色葡萄球菌最有效毒素之一的強大免疫應答。在任意的實施方案中，LukA變異多肽是全長LukA蛋白的變體，所述全長LukA蛋白包含對應於全長成熟LukA蛋白序列的所有氨基酸殘基。本文所指的“成熟”殺白細胞素蛋白序列是缺乏氨基末端分泌信號的殺白細胞素蛋白序列，其通常包含氨基末端的前27-28個氨基酸殘基。 A first aspect of the invention relates to variants, ie non-naturally occurring Staphylococcus aureus ( S. aureus ) leukocidin A (LukA) proteins or polypeptides. These mutated LukA proteins or polypeptides contain insertions, substitutions and/or deletions of one or more amino acid residues, making the LukAB two-component toxin non-cytotoxic, stabilizing the LukAB heterodimer, increasing the melting temperature, and/or increasing Solubility. As described herein, these variant LukA proteins and polypeptides are ideal vaccine antigen candidates and can be administered alone or in combination with leukocidin B (LukB) wild-type or variant proteins or polypeptides. When administered in combination with the LukB protein or its polypeptides, the resulting toxoid mimics the structure of the S. aureus LukB toxin, thereby promoting a robust immune response against one of S. aureus' most potent toxins. In any of the embodiments, the LukA variant polypeptide is a variant of the full-length LukA protein comprising all amino acid residues corresponding to the sequence of the full-length mature LukA protein. A "mature" leukocidin protein sequence, as referred to herein, is a leukocidin protein sequence lacking the amino-terminal secretion signal, which typically comprises the first 27-28 amino acid residues at the amino-terminus.

在任意的實施方案中，LukA變異多肽是小於全長成熟LukA蛋白的變體。在任意的實施方案中，LukA變異多肽長度至少為100個氨基酸殘基。在任意的實施方案中，LukA變異多肽是至少110、至少120、至少130、至少140、至少150、至少160、至少170、至少180、至少190、至少200、至少210、至少220、至少230、至少240、至少250、至少260、至少270、至少280、至少290、至少300個氨基酸殘基的長度。In any of the embodiments, the LukA variant polypeptide is a variant that is less than the full length mature LukA protein. In any embodiment, the LukA variant polypeptide is at least 100 amino acid residues in length. In any embodiment, the LukA variant polypeptide is at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, At least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300 amino acid residues in length.

雖然本文描述的示例性LukA變異蛋白和多肽是克隆複合物CC8 (SEQ ID NO:1)和CC45 (SEQ ID NO:2)變異的LukA蛋白(見下表1)，但是本領域技術人員將容易理解，鑒定LukA的氨基酸取代和/或缺失所基於的SEQ ID NO:1和SEQ ID NO:2是在各種克隆複合物中保守的氨基酸殘基，或者是在各種克隆複合物中高度保守的LukA區域內的氨基酸殘基。事實上，來自15個不同金黃色葡萄球菌菌株的LukA蛋白序列的比對(見圖1)顯示，本文中鑒定為易變異殘基的氨基酸殘基是在所有15個比對的LukA氨基酸序列中保守的殘基。雖然所鑒定的變異殘基的位置在單個LukA序列之間可能不同，但序列比對顯示了這些位置之間的對應關系。為了清楚起見，從序列比對中產生具有SEQ ID NO:25的氨基酸序列的LukA共有序列，並用於指定特定氨基酸變異的位置。例如，SEQ ID NO:25中賴氨酸殘基83位的氨基酸取代對應於SEQ ID NO:1的LukA序列中80位的賴氨酸殘基，對應於SEQ ID NO:2的LukA序列中81位的賴氨酸殘基，以及對應於SEQ ID NO:26–38的LukA序列中83位的賴氨酸殘基。因此，本文所述的經鑒定的氨基酸變體可以普遍應用於現在已知的或將來的任何LukA氨基酸序列的相應氨基酸殘基。Although the exemplary LukA variant proteins and polypeptides described herein are variant LukA proteins of cloning complexes CC8 (SEQ ID NO:1) and CC45 (SEQ ID NO:2) (see Table 1 below), those skilled in the art will readily It is understood that the identification of amino acid substitutions and/or deletions of LukA based on SEQ ID NO: 1 and SEQ ID NO: 2 are amino acid residues that are conserved in various clonal complexes, or that LukA is highly conserved in various clonal complexes Amino acid residues in the region. In fact, an alignment of LukA protein sequences from 15 different S. aureus strains (see Figure 1) revealed that the amino acid residues identified herein as easily variable residues were present in all 15 aligned LukA amino acid sequences Conserved residues. Although the positions of the identified variant residues may differ between individual LukA sequences, the sequence alignment shows a correspondence between these positions. For clarity, a LukA consensus sequence having the amino acid sequence of SEQ ID NO: 25 was generated from the sequence alignment and used to designate the positions of specific amino acid variations. For example, the amino acid substitution at position 83 of the lysine residue in SEQ ID NO:25 corresponds to the lysine residue at position 80 in the LukA sequence of SEQ ID NO:1, and corresponds to the 81 position in the LukA sequence of SEQ ID NO:2. The lysine residue at position , and the lysine residue at position 83 in the LukA sequence corresponding to SEQ ID NO:26-38. Thus, the identified amino acid variants described herein can be generally applied to the corresponding amino acid residues of any LukA amino acid sequence, now known or in the future.

根據本發明的這個方面，在任意的實施方案中，LukA變異多肽包含對應於SEQ ID NO:25的殘基Lys83、Ser141、Val113、Val193的一個或多個氨基酸殘基上的氨基酸殘基***、取代和/或缺失。在任意的實施方案中，除了上述一個或多個氨基酸殘基***、取代和/或缺失之外，LukA變異多肽還包含對應於SEQ ID NO:25的Glu323的氨基酸殘基上的氨基酸取代或缺失。在任意的實施方案中，在Glu323的氨基酸取代或缺失包括在SEQ ID NO:25的323位(Glu323Ala)的穀氨酸到丙氨酸的取代。According to this aspect of the invention, in any embodiment, the LukA variant polypeptide comprises amino acid residue insertions corresponding to one or more amino acid residues of Lys83, Ser141, Val113, Val193 of residues Lys83, Ser141, Val113, Val193 of SEQ ID NO:25, substitutions and/or deletions. In any embodiment, in addition to the above-mentioned one or more amino acid residue insertions, substitutions and/or deletions, the LukA variant polypeptide also comprises amino acid substitutions or deletions corresponding to the amino acid residues of Glu323 of SEQ ID NO:25 . In any of the embodiments, the amino acid substitution or deletion at Glu323 comprises a glutamic acid to alanine substitution at position 323 of SEQ ID NO:25 (Glu323Ala).

在任意的實施方案中，在LukA(以及本文所述的LukB)的一個或多個鑒定位置的氨基酸取代是保守取代。這種保守的取代包括用一個氨基酸殘基替換另一個同一類型的氨基酸殘基，這種替換作為功能等同物，導致無聲的改變。也就是說，相對於天然序列的變化不會明顯降低LukA的基本性質。這些種類的氨基酸殘基包括非極性(疏水性)氨基酸(例如丙氨酸、亮氨酸、異亮氨酸、纈氨酸、脯氨酸、苯丙氨酸、色氨酸和甲硫氨酸)；極性中性氨基酸(例如甘氨酸、絲氨酸、蘇氨酸、半胱氨酸、酪氨酸、天冬醯胺和穀氨醯胺)；帶正電荷的(鹼性)氨基酸(如精氨酸、賴氨酸和組氨酸)；和帶負電荷的(酸性)氨基酸(例如天冬氨酸和穀氨酸)。In any of the embodiments, the amino acid substitutions at one or more of the identified positions in LukA (and LukB as described herein) are conservative substitutions. Such conservative substitutions involve the substitution of one amino acid residue for another of the same type, which acts as a functional equivalent, resulting in a silent change. That is, changes relative to the native sequence do not significantly reduce the essential properties of LukA. These classes of amino acid residues include nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine ); polar neutral amino acids (such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine); positively charged (basic) amino acids (such as arginine , lysine and histidine); and negatively charged (acidic) amino acids (such as aspartic acid and glutamic acid).

在其他實施方案中，本文所述的變異殺白細胞素多肽的一個或多個鑒定位置的氨基酸取代是非保守性改變(即破壞鑒定區域的序列、結構、功能或活性的取代)。為了減少或減輕蛋白質的細胞毒性，這種取代可能是理想的。非保守取代包括用不同種類的氨基酸殘基取代一個特定種類的氨基酸殘基。例如，用極性中性氨基酸取代非極性(疏水性)氨基酸殘基，反之亦然。在另一個實施方案中，非保守取代包括帶正電荷(鹼性)氨基酸殘基被帶負電荷(酸性)氨基酸殘基（例如天冬氨酸和穀氨酸）取代，反之亦然。這種分子改變可以通過本領域熟知的方法完成，包括使用單鏈模板在質粒模板上進行引物延伸(Kunkel等人，Proc. Acad. Sci., USA 82:488-492 (1985), which is hereby incorporated by reference in its entirety，在本文中通過全文引入作為參考)，雙鏈DNA模板(Papworth等人，Strategies 9(3):3-4 (1996)，在本文中通過全文引入作為參考)，和通過PCR克隆(Braman，J. (ed.), IN VITRO MUTAGENESIS PROTOCOLS, 2nd ed. Humana Press, Totowa, N.J. (2002)，其全部內容通過引用結合於此)。In other embodiments, the amino acid substitutions at one or more of the identified positions of the variant leukocidin polypeptides described herein are non-conservative changes (ie, substitutions that disrupt the sequence, structure, function, or activity of the identified regions). Such substitutions may be desirable in order to reduce or lessen the cytotoxicity of the protein. Non-conservative substitutions involve replacing an amino acid residue of a particular class with an amino acid residue of a different class. For example, a polar neutral amino acid is substituted for a non-polar (hydrophobic) amino acid residue and vice versa. In another embodiment, non-conservative substitutions include the substitution of positively charged (basic) amino acid residues with negatively charged (acidic) amino acid residues (eg, aspartic acid and glutamic acid), and vice versa. Such molecular alterations can be accomplished by methods well known in the art, including primer extension on plasmid templates using single-stranded templates (Kunkel et al., Proc. Acad. Sci., USA 82:488-492 (1985), which is hereby incorporated by reference in its entirety, incorporated by reference in its entirety herein), a double-stranded DNA template (Papworth et al., Strategies 9(3):3-4 (1996), incorporated by reference herein in its entirety), and Cloning by PCR (Braman, J. (ed.), IN VITRO MUTAGENESIS PROTOCOLS, 2nd ed. Humana Press, Totowa, N.J. (2002), the entire contents of which are hereby incorporated by reference).

在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:25的83位(Lys83Met)賴氨酸的殘基上包含賴氨酸到甲硫氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:1的80位(Lys80Met)賴氨酸的殘基上包含賴氨酸到甲硫氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽包含在對應於SEQ ID NO:2的81位(Lys81Met)賴氨酸的殘基上包含賴氨酸到甲硫氨酸的取代。In any embodiment, the LukA variant polypeptide of the present invention comprises a substitution of lysine to methionine at the residue corresponding to lysine at position 83 (Lys83Met) of SEQ ID NO:25. In any embodiment, the LukA variant polypeptide of the present invention comprises a substitution of lysine to methionine at the residue corresponding to lysine at position 80 (Lys80Met) of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide of the invention comprises a substitution of lysine to methionine at a residue corresponding to lysine at position 81 (Lys81Met) of SEQ ID NO:2.

在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:25的141位(Ser141Ala)的絲氨酸的殘基上包含絲氨酸到丙氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:1的138位(Ser138Ala)的絲氨酸的殘基上包含絲氨酸到丙氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:2的139位(Ser139Ala)的絲氨酸的殘基上包含絲氨酸到丙氨酸的取代。In any embodiment, the LukA variant polypeptide of the invention comprises a serine to alanine substitution at the residue corresponding to serine at position 141 (Ser141Ala) of SEQ ID NO:25. In any embodiment, the LukA variant polypeptide of the invention comprises a serine to alanine substitution at the residue corresponding to serine at position 138 (Ser138Ala) of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide of the invention comprises a serine to alanine substitution at the residue corresponding to serine at position 139 (Ser139Ala) of SEQ ID NO:2.

在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:25的113 位(Val113Ile)的纈氨酸的殘基上包含纈氨酸到異亮氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:1的110 位(Val110Ile)的纈氨酸的殘基上包含纈氨酸到異亮氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:2的111位 (Val111Ile)的纈氨酸的殘基上包含纈氨酸到異亮氨酸的取代。In any embodiment, the LukA variant polypeptide of the invention comprises a substitution of valine to isoleucine at the residue corresponding to valine at position 113 (Val113Ile) of SEQ ID NO:25. In any embodiment, the LukA variant polypeptide of the present invention comprises a substitution of valine to isoleucine at the residue corresponding to valine at position 110 (Val110Ile) of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide of the invention comprises a substitution of valine to isoleucine at a residue corresponding to valine at position 111 (Val111Ile) of SEQ ID NO:2.

在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:25的193 位(Val193Ile)的纈氨酸的殘基上包含纈氨酸到異亮氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:1的190 位(Val190Ile)的纈氨酸的殘基上包含纈氨酸到異亮氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽在對應於SEQ ID NO:2的191位 (Val191Ile)的纈氨酸的殘基上包含纈氨酸到異亮氨酸的取代。In any embodiment, the LukA variant polypeptide of the invention comprises a substitution of valine to isoleucine at the residue corresponding to valine at position 193 (Val193Ile) of SEQ ID NO:25. In any embodiment, the LukA variant polypeptide of the present invention comprises a substitution of valine to isoleucine at the residue corresponding to valine at position 190 (Val190Ile) of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide of the present invention comprises a substitution of valine to isoleucine at a residue corresponding to valine at position 191 (Val191Ile) of SEQ ID NO:2.

在任意的實施方案中，本發明的LukA變異多肽除了對應於SEQ ID NO:25的Lys83、Ser141、Val113和Val193的殘基上的任何一個或多個取代之外，還包含對應於SEQ ID NO:25的穀氨酸殘基323 位(Glu323Ala)的殘基上的穀氨酸到丙氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽除了對應於SEQ ID NO:1的Lys80、Ser138、Val110、Val190的殘基上的任何一個或多個取代之外，還包含對應於SEQ ID NO:1的穀氨酸殘基320 位(Glu320Ala)的殘基上的穀氨酸到丙氨酸的取代。在任意的實施方案中，本發明的LukA變異多肽除了對應於SEQ ID NO:25的Lys81、Ser139、Val111、Val191的殘基上的任何一個或多個取代之外，還包含對應於SEQ ID NO:2的穀氨酸殘基321位 (Glu321Ala)的殘基上的穀氨酸到丙氨酸的取代。In any embodiment, the LukA variant polypeptide of the present invention, in addition to any one or more substitutions corresponding to residues of Lys83, Ser141, Val113 and Val193 of SEQ ID NO: 25, also comprises a residue corresponding to SEQ ID NO: 25. Glutamic acid to alanine substitution at residue 323 of the glutamic acid residue of :25 (Glu323Ala). In any embodiment, the LukA variant polypeptide of the present invention, in addition to any one or more substitutions on the residues corresponding to Lys80, Ser138, Val110, Val190 of SEQ ID NO: 1, also comprises a residue corresponding to SEQ ID NO: Glutamate to alanine substitution on residue 320 of the glutamic acid residue of :1 (Glu320Ala). In any embodiment, the LukA variant polypeptide of the present invention, in addition to any one or more substitutions on the residues corresponding to Lys81, Ser139, Val111, Val191 of SEQ ID NO: 25, also comprises a residue corresponding to SEQ ID NO: 25 Substitution of glutamic acid to alanine at the residue at position 321 of the glutamic acid residue of :2 (Glu321Ala).

在任意的實施方案中，本發明的LukA變異多肽包含對應於SEQ ID NO:25的Lys83、Ser141、Val113和Val193的兩個前述氨基酸殘基上具有氨基酸殘基***、取代和/或缺失的多肽。在任意的實施方案中，LukA變異多肽包含在三個前述氨基酸殘基上的氨基酸殘基***、取代和/或缺失。在任意的實施方案中，LukA變異多肽包含在所有四個前述氨基酸殘基上的氨基酸殘基***、取代和/或缺失。在任意的實施方案中，LukA變異多肽在對應於SEQ ID NO:25的Lys83Met、Ser141Ala、Val113Ile和Val193Ile的上述氨基酸殘基上包含賴氨酸到甲硫氨酸、絲氨酸到丙氨酸和纈氨酸到異亮氨酸的氨基酸取代。在任意的實施方案中，LukA變異多肽進一步包括對應於SEQ ID NO:25的殘基323位 (Glu323Ala)的氨基酸殘基處穀氨酸到丙氨酸的氨基酸取代，即變異LukA包括對應於SEQ ID NO:25的Lys83Met、Ser141Ala、Val113Ile、Val193Ile和Glu323Ala的取代。In any embodiment, the LukA variant polypeptide of the present invention comprises a polypeptide having amino acid residue insertion, substitution and/or deletion on the two aforementioned amino acid residues corresponding to Lys83, Ser141, Val113 and Val193 of SEQ ID NO:25 . In any embodiment, the LukA variant polypeptide comprises amino acid residue insertions, substitutions and/or deletions at the three aforementioned amino acid residues. In any embodiment, the LukA variant polypeptide comprises amino acid residue insertions, substitutions and/or deletions at all four of the aforementioned amino acid residues. In any embodiment, the LukA variant polypeptide comprises lysine to methionine, serine to alanine and valine at the above amino acid residues corresponding to Lys83Met, Ser141Ala, Val113Ile and Val193Ile of SEQ ID NO:25 Acid to Isoleucine Amino Acid Substitution. In any embodiment, the LukA variant polypeptide further comprises an amino acid substitution from glutamic acid to alanine at the amino acid residue corresponding to residue 323 (Glu323Ala) of SEQ ID NO: 25, that is, the variant LukA comprises amino acid substitutions corresponding to SEQ ID NO:25. Substitutions of Lys83Met, Ser141Ala, Val113Ile, Val193Ile and Glu323Ala of ID NO:25.

本發明的示例性變異LukA多肽具有對應於SEQ ID NO:25中的Lys83Met、Ser141Ala、Val113Ile、Val193Ile和Glu323Ala的氨基酸取代。在任意的實施方案中，LukA變異多肽是CC8 LukA變體，其包含對應於SEQ ID NO:1中Lys80Met、Ser138Ala、Val110Ile、Val190Ile和Glu320Ala的氨基酸取代。在任意的實施方案中，該LukA變異多肽具有SEQ ID NO:3的氨基酸序列，或具有與SEQ ID NO:3的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。Exemplary variant LukA polypeptides of the invention have amino acid substitutions corresponding to Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala in SEQ ID NO:25. In any embodiment, the LukA variant polypeptide is a CC8 LukA variant comprising amino acid substitutions corresponding to Lys80Met, Ser138Ala, Val110Ile, Val190Ile, and Glu320Ala in SEQ ID NO:1. In any embodiment, the LukA variant polypeptide has the amino acid sequence of SEQ ID NO: 3, or has at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the amino acid sequence of SEQ ID NO: 3 Amino acid sequences with % sequence similarity.

在任意的實施方案中，LukA變異多肽是CC45 LukA變異多肽，其包含對應於SEQ ID NO:2中Lys81Met、Ser139Ala、Val111Ile、Val191Ile和Glu321Ala的氨基酸取代。在任意的實施方案中，該LukA變異多肽具有SEQ ID NO:4的氨基酸序列，或具有與SEQ ID NO:4的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。其他示例性的LukA變異多肽包括SEQ ID NO:26-38的LukA蛋白質中的任何一種，其包含對應於SEQ ID NO:25中Lys83Met、Ser141Ala、Val113Ile、Val193Ile和Glu323Ala的取代的氨基酸取代。In any embodiment, the LukA variant polypeptide is a CC45 LukA variant polypeptide comprising amino acid substitutions corresponding to Lys81Met, Ser139Ala, Val111Ile, Val191Ile, and Glu321Ala in SEQ ID NO:2. In any embodiment, the LukA variant polypeptide has the amino acid sequence of SEQ ID NO: 4, or has at least 85%, at least 90%, at least 95%, at least 97% or at least 99% of the amino acid sequence of SEQ ID NO: 4 Amino acid sequences with % sequence similarity. Other exemplary LukA variant polypeptides include any of the LukA proteins of SEQ ID NOs: 26-38 comprising amino acid substitutions corresponding to the substitutions of Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala in SEQ ID NO:25.

在任意的實施方案中，本文所述的LukA變異多肽包含對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的一個或多個氨基酸殘基上的氨基酸取代。在一個實施方案中，在一個或多個前述殘基上的氨基酸取代引入了能夠形成二硫鍵的半胱氨酸殘基，以穩定LukAB異二聚體結構的構象。例如，在一個實施方案中，本文所述的LukA變異多肽在對應於SEQ ID NO:25的Tyr74 (Tyr74Cys)的氨基酸殘基上包含酪氨酸至半胱氨酸的取代，並且在對應於SEQ ID NO:25的Asp140 (Asp140Cys)的氨基酸殘基上包含天冬醯胺至半胱氨酸的取代。74位和140位處的這些半胱氨酸殘基形成二硫鍵，從而相對於野生型LukA或相對於不含能形成二硫鍵的成對半胱氨酸殘基的其他變異LukA多肽，增加了變異LukA的熱穩定性。In any embodiment, the LukA variant polypeptide described herein comprises an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO:25. In one embodiment, amino acid substitutions at one or more of the foregoing residues introduce cysteine residues capable of forming disulfide bonds to stabilize the conformation of the LukAB heterodimer structure. For example, in one embodiment, the LukA variant polypeptide described herein comprises a substitution of tyrosine to cysteine at an amino acid residue corresponding to Tyr74 (Tyr74Cys) of SEQ ID NO:25, and a substitution at an amino acid residue corresponding to SEQ ID NO:25 The amino acid residue of Aspl40 (Asp140Cys) of ID NO:25 contains an asparagine to cysteine substitution. These cysteine residues at positions 74 and 140 form disulfide bonds, so that relative to wild-type LukA or relative to other variant LukA polypeptides that do not contain a pair of cysteine residues capable of forming a disulfide bond, Increased thermostability of mutant LukA.

在另一個實施方案中，本文所述的LukA變異多肽在對應於SEQ ID NO:25的Gly149 (Gly149Cys)的氨基酸殘基上包含甘氨酸到半胱氨酸的取代，並且在對應於SEQ ID NO:25的Gly156 (Gly156Cys)的氨基酸殘基上包含甘氨酸到半胱氨酸的取代。這些在149和156位引入的半胱氨酸殘基形成二硫鍵，從而相對於野生型LukA或相對於不含能形成二硫鍵的成對半胱氨酸殘基的其他變異LukA多肽增加了變異LukA的熱穩定性。In another embodiment, the LukA variant polypeptide described herein comprises a glycine to cysteine substitution at an amino acid residue corresponding to Gly149 (Gly149Cys) of SEQ ID NO: 25, and at an amino acid residue corresponding to SEQ ID NO: 25: Gly156 of 25 (Gly156Cys) contains a glycine to cysteine substitution at the amino acid residue. These introduced cysteine residues at positions 149 and 156 form disulfide bonds, resulting in increased The thermostability of mutant LukA was determined.

在任意的實施方案中，LukA變異多肽在對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，在任一氨基酸殘基上的氨基酸取代都包括如上所述的半胱氨酸殘基的引入。在任意的實施方案中，LukA變異多肽在對應於SEQ ID NO:1的氨基酸殘基Tyr71、Asp137、Gly146和Gly153的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，任一氨基酸殘基上的氨基酸取代都包括引入半胱氨酸殘基。在任意的實施方案中，LukA變異多肽在對應於SEQ ID NO:2的氨基酸殘基Tyr72、Asp138、Gly147和Gly154的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，任一氨基酸殘基上的氨基酸取代都包括引入半胱氨酸殘基。In any of the embodiments, the LukA variant polypeptide comprises an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149 and Gly156 of SEQ ID NO:25. In any of the embodiments, amino acid substitutions at any amino acid residue include the introduction of a cysteine residue as described above. In any of the embodiments, the LukA variant polypeptide comprises an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Tyr71, Asp137, Gly146 and Gly153 of SEQ ID NO:1. In any of the embodiments, amino acid substitutions at any amino acid residue include the introduction of a cysteine residue. In any of the embodiments, the LukA variant polypeptide comprises an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Tyr72, Asp138, Gly147 and Gly154 of SEQ ID NO:2. In any of the embodiments, amino acid substitutions at any amino acid residue include the introduction of a cysteine residue.

在任意的實施方案中，LukA變異多肽包含在對應於Lys83、Ser141、Val113、Val193和Glu323的一個或多個氨基酸殘基上的氨基酸取代，以及在對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的一個或多個氨基酸殘基上的氨基酸取代。在任意的實施方案中，LukA變異多肽包含對應於SEQ ID NO:25的殘基Lys83、Ser141、Val113、Val193和Glu323以及殘基Tyr74、Asp140、Gly149和Gly156的氨基酸殘基上的氨基酸取代。In any embodiment, the LukA variant polypeptide comprises amino acid substitutions at one or more amino acid residues corresponding to Lys83, Ser141, Val113, Val193, and Glu323, and at amino acid residue Tyr74 corresponding to SEQ ID NO:25 Amino acid substitutions at one or more amino acid residues of , Asp140, Gly149 and Gly156. In any embodiment, the LukA variant polypeptide comprises amino acid substitutions at amino acid residues corresponding to residues Lys83, Ser141 , Val113, Val193 and Glu323 and residues Tyr74, Aspl40, Gly149 and Gly156 of SEQ ID NO:25.

在任意的實施方案中，示例性的LukA變異多肽是CC8 LukA變異多肽，其在對應於SEQ ID NO:1的Lys80、Ser138、Val110、Val190、Glu320、Tyr71、Asp137、Gly146和Gly153中每一個的殘基上具有氨基酸取代。在任意的實施方案中，示例性的LukA變異多肽是CC8 LukA變異多肽，其在對應於SEQ ID NO:1的Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Glu320Ala、Tyr71Cys、Asp137Cys、Gly146Cys和Gly153Cys中每一個的殘基上具有氨基酸取代。在任意的實施方案中，該CC8 LukA變異多肽包含SEQ ID NO:5的氨基酸序列，或具有與SEQ ID NO:5的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, the exemplary LukA variant polypeptide is a CC8 LukA variant polypeptide at Lys80, Ser138, Val110, Val190, Glu320, Tyr71, Asp137, Gly146 and Gly153 corresponding to each of SEQ ID NO:1 Amino acid substitutions on residues. In any embodiment, the exemplary LukA variant polypeptide is a CC8 LukA variant polypeptide in which each of Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Glu320Ala, Tyr71Cys, Asp137Cys, Gly146Cys, and Gly153Cys corresponding to SEQ ID NO:1 Amino acid substitutions on residues. In any embodiment, the CC8 LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:5, or has at least 85%, at least 90%, at least 95%, at least 97%, or at least Amino acid sequences with 99% sequence similarity.

在任意的實施方案中，示例性的LukA變異多肽是CC45 LukA變異多肽，其在對應於SEQ ID NO:2的Lys81、Ser139、Val111、Val191、Glu321、Tyr72、Asp138、Gly147和Gly154中每一個的殘基上具有氨基酸取代。在任意的實施方案中，示例性的LukA變異多肽是CC45 LukA變異多肽，其在對應於SEQ ID NO:2的Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Glu321Ala、Tyr72Cys、Asp138Cys、Gly147Cys和Gly154Cys中每一個的殘基上具有氨基酸取代。在任意的實施方案中，該CC45 LukA變異多肽包含SEQ ID NO:6的氨基酸序列，或具有與SEQ ID NO:6的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, the exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide in which each of Lys81, Ser139, Val111, Val191, Glu321, Tyr72, Asp138, Gly147, and Gly154 corresponding to SEQ ID NO:2 Amino acid substitutions on residues. In any embodiment, the exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide having Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Glu321Ala, Tyr72Cys, Asp138Cys, Gly147Cys, and Gly154Cys corresponding to each of SEQ ID NO:2. Amino acid substitutions on residues. In any embodiment, the CC45 LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:6, or has at least 85%, at least 90%, at least 95%, at least 97%, or at least Amino acid sequences with 99% sequence similarity.

其他示例性的LukA變異多肽包括SEQ ID NO:26-38的LukA蛋白質中的任何一種，其包含對應於SEQ ID NO:25的Lys83Met、Ser141Ala、Val113Ile、Val193Ile、Glu323、Tyr74、Asp140、Gly149和Gly156的氨基酸取代。Other exemplary LukA variant polypeptides include any of the LukA proteins of SEQ ID NO:26-38 comprising Lys83Met, Ser141Ala, Val113Ile, Val193Ile, Glu323, Tyr74, Aspl40, Gly149, and Gly156 corresponding to SEQ ID NO:25 amino acid substitutions.

在任意的實施方案中，本文所述的LukA變異多肽在對應於SEQ ID NO:25的氨基酸殘基Thr249的氨基酸殘基上包含氨基酸取代或缺失。在任意的實施方案中，LukA變體包含在對應於Thr249的殘基上的取代，其中所述取代是在該殘基(Thr249Val)上蘇氨酸到纈氨酸的取代。在任意的實施方案中，本文所述的LukA變異多肽在對應於SEQ ID NO:1的氨基酸殘基Thr246的氨基酸殘基上包含氨基酸取代或缺失。在任意的實施方案中，本文所述的LukA變異多肽在對應於SEQ ID NO:2的氨基酸殘基Thr247的氨基酸殘基上包含氨基酸取代或缺失。In any embodiment, the LukA variant polypeptide described herein comprises an amino acid substitution or deletion at an amino acid residue corresponding to amino acid residue Thr249 of SEQ ID NO:25. In any of the embodiments, the LukA variant comprises a substitution at the residue corresponding to Thr249, wherein said substitution is a threonine to valine substitution at this residue (Thr249Val). In any embodiment, the LukA variant polypeptide described herein comprises an amino acid substitution or deletion at an amino acid residue corresponding to amino acid residue Thr246 of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide described herein comprises an amino acid substitution or deletion at an amino acid residue corresponding to amino acid residue Thr247 of SEQ ID NO:2.

在任意的實施方案中，本文描述的LukA變異多肽包含在對應於SEQ ID NO:25的Thr249的氨基酸殘基上的氨基酸取代，與本文描述的任何其他氨基酸殘基取代相結合，即在對應於SEQ ID NO:25的Lys83、Ser141、Val113、Val193、Glu323、Tyr74、Asp140、Gly149和Gly156的殘基上的取代。在任意的實施方案中，本文所述的LukA變異多肽包含對應於SEQ ID NO:25的Thr249的氨基酸殘基上的氨基酸取代，以及本文所述的至少兩個、至少三個、至少四個、至少五個、至少六個、至少七個、至少八個或所有九個其他氨基酸殘基取代。在任意的實施方案中，LukA變異多肽在對應於SEQ ID NO:25的Lys83、Ser141、Val113、Val193、Glu323和Thr249的每個殘基上包含氨基酸取代。In any embodiment, the LukA variant polypeptide described herein comprises an amino acid substitution at the amino acid residue corresponding to Thr249 of SEQ ID NO: 25, in combination with any other amino acid residue substitution described herein, i.e. at the amino acid residue corresponding to Substitutions at residues of Lys83, Ser141, Val113, Val193, Glu323, Tyr74, Aspl40, Gly149 and Gly156 of SEQ ID NO:25. In any embodiment, the LukA variant polypeptide described herein comprises an amino acid substitution corresponding to an amino acid residue of Thr249 of SEQ ID NO: 25, and at least two, at least three, at least four, At least five, at least six, at least seven, at least eight or all nine other amino acid residues are substituted. In any of the embodiments, the LukA variant polypeptide comprises an amino acid substitution at each of the residues corresponding to Lys83, Ser141, Val113, Val193, Glu323 and Thr249 of SEQ ID NO:25.

在任意的實施方案中，示例性的LuCA變異多肽是CC8 LukA變異多肽，其在對應於SEQ ID NO:1的Lys80、Ser138、Val110、Val190、Glu320和Thr246中每一個的殘基上具有氨基酸取代。在任意的實施方案中，示例性的LukA變異多肽是CC8 LukA變異多肽，其在對應於SEQ ID NO:1的Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Glu320Ala和Thr246Val中每一個的殘基上具有氨基酸取代。在一個實施方案中，在對應於上述每個位置的殘基上具有氨基酸取代的示例性的LukA變異多肽具有SEQ ID NO:7的氨基酸序列，或具有與SEQ ID NO:7的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, the exemplary LuCA variant polypeptide is a CC8 LukA variant polypeptide having amino acid substitutions at residues corresponding to each of Lys80, Ser138, Val110, Val190, Glu320, and Thr246 of SEQ ID NO:1 . In any embodiment, the exemplary LukA variant polypeptide is a CC8 LukA variant polypeptide having amino acid substitutions at residues corresponding to each of Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Glu320Ala, and Thr246Val of SEQ ID NO:1 . In one embodiment, the exemplary LukA variant polypeptide having amino acid substitutions at the residues corresponding to each of the above positions has the amino acid sequence of SEQ ID NO: 7, or has an amino acid sequence at least 855 from the amino acid sequence of SEQ ID NO: 7 %, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to amino acid sequences.

在任意的實施方案中，示例性的LukA變異多肽是CC45 LukA變異多肽，其在對應於SEQ ID NO:2的Lys81、Ser139、Val111、Val191、Glu321和Thr247中每一個的殘基上具有氨基酸取代。在任意的實施方案中，示例性的LukA變異多肽是CC45 LukA變異多肽，其在對應於SEQ ID NO:2的Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Glu321Ala和Thr247Val中每一個的殘基上具有氨基酸取代。在一個實施方案中，在對應於上述每個位置的殘基上具有氨基酸取代的示例性的LukA變異多肽具有SEQ ID NO:8的氨基酸序列，或具有與SEQ ID NO:8的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, the exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide having amino acid substitutions at residues corresponding to each of Lys81, Ser139, Val111, Val191, Glu321, and Thr247 of SEQ ID NO:2 . In any embodiment, an exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide having amino acid substitutions at residues corresponding to each of Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Glu321Ala, and Thr247Val of SEQ ID NO:2 . In one embodiment, an exemplary LukA variant polypeptide having amino acid substitutions at residues corresponding to each of the above positions has the amino acid sequence of SEQ ID NO: 8, or has an amino acid sequence at least 855 from the amino acid sequence of SEQ ID NO: 8 %, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to amino acid sequences.

其他示例性的LukA變異多肽包括SEQ ID NO:26-38的LukA蛋白中的任何一種，其在對應於SEQ ID NO:25的Lys83、Ser141、Val113、Val193、Glu323和Thr249的氨基酸殘基上包含所述氨基酸取代。Other exemplary LukA variant polypeptides include any of the LukA proteins of SEQ ID NOs: 26-38 comprising amino acid residues corresponding to Lys83, Ser141, Val113, Val193, Glu323 and Thr249 of SEQ ID NO: 25 The amino acid substitution.

在任意的實施方案中，LukA變異多肽在對應於SEQ ID NO:25的Lys83、Ser141、Val113、Val193、Glu323、Thr249、Tyr74、Asp140、Gly149和Gly156的每個殘基上包含氨基酸取代。In any of the embodiments, the LukA variant polypeptide comprises an amino acid substitution at each of the residues corresponding to Lys83, Ser141, Val113, Val193, Glu323, Thr249, Tyr74, Aspl40, Gly149, and Gly156 of SEQ ID NO:25.

在任意的實施方案中，示例性的LukA變異多肽是CC8 LukA變異多肽，其在對應於SEQ ID NO:1的Lys80、Ser138、Val110、Val190、Glu320、Tyr71、Asp137、Gly146、Gly153和Thr246中每一個的殘基上具有氨基酸取代。在任意的實施方案中，示例性的LukA變異多肽是CC8 LukA變異多肽，其在對應於SEQ ID NO:1的Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Glu320Ala、Tyr71Cys、Asp137Cys、Gly146Cys、Gly153Cys和Thr246Val中每一個的殘基上具有氨基酸取代。在一個實施方案中，在對應於上述每個位置的殘基上具有氨基酸取代的示例性的LukA變異多肽具有SEQ ID NO:9的氨基酸序列，或具有與SEQ ID NO:9的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, the exemplary LukA variant polypeptide is a CC8 LukA variant polypeptide, each of which corresponds to Lys80, Ser138, Val110, Val190, Glu320, Tyr71, Asp137, Gly146, Gly153 and Thr246 of SEQ ID NO:1 One has amino acid substitutions on residues. In any embodiment, the exemplary LukA variant polypeptide is a CC8 LukA variant polypeptide, each of which corresponds to Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Glu320Ala, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys and Thr246Val of SEQ ID NO:1 One has amino acid substitutions on residues. In one embodiment, an exemplary LukA variant polypeptide having amino acid substitutions at residues corresponding to each of the above positions has the amino acid sequence of SEQ ID NO: 9, or has an amino acid sequence at least 855 from the amino acid sequence of SEQ ID NO: 9. %, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to amino acid sequences.

在任意的實施方案中，示例性的LukA變異多肽是CC45 LukA變異多肽，其在對應於SEQ ID NO:2的Lys81、Ser139、Val111、Val191、Glu321、Tyr72、Asp138、Gly147、Gly154和Thr247中每一個的殘基上具有氨基酸取代。在任意的實施方案中，示例性的LukA變異多肽是CC45 LukA變異多肽，其在對應於SEQ ID NO:2的Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Glu321Ala、Tyr72Cys、Asp138Cys、Gly147Cys、Gly154Cys和Thr247Ala中每一個的殘基上具有氨基酸取代。在一個實施方案中，在對應於上述每個位置的殘基上具有氨基酸取代的示例性的LukA變異多肽具有SEQ ID NO:10的氨基酸序列，或具有與SEQ ID NO:10的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, the exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide, each of which corresponds to Lys81, Ser139, Val111, Val191, Glu321, Tyr72, Asp138, Gly147, Gly154, and Thr247 of SEQ ID NO:2. One has amino acid substitutions on residues. In any embodiment, the exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide, each of which corresponds to Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Glu321Ala, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys, and Thr247Ala of SEQ ID NO:2. One has amino acid substitutions on residues. In one embodiment, the exemplary LukA variant polypeptide having amino acid substitutions at the residues corresponding to each of the above positions has the amino acid sequence of SEQ ID NO: 10, or has an amino acid sequence at least 85 times the amino acid sequence of SEQ ID NO: 10 %, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to amino acid sequences.

其他示例性的LukA變異多肽包括SEQ ID NO:26-38的LukA蛋白質中的任一種，其在對應於SEQ ID NO:25的Lys83、Ser141、Val113、Val193、Glu323、Thr249、Tyr74、Asp140、Gly149和Gly156的殘基上包含所述氨基酸取代。Other exemplary LukA variant polypeptides include any of the LukA proteins of SEQ ID NOs: 26-38 at Lys83, Ser141, Val113, Val193, Glu323, Thr249, Tyr74, Aspl40, Gly149 corresponding to SEQ ID NO:25 and Gly156 residues comprising the amino acid substitutions.

下表1提供了本文公開的示例性的變異LukA氨基酸序列。Table 1 below provides exemplary variant LukA amino acid sequences disclosed herein.

表surface 1.1. 示例性Exemplary LukALuka 多肽氨基酸序列Peptide Amino Acid Sequence SEQ ID NOSEQ ID NO 名稱name 描述describe 1 1 LukA CC8 WTLukA CC8 WT HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHKEEKNSNWLKYPSEYHVDFQVKRNRKTEILDQLPKNKISTAKVDSTFSYSSGGKFDSTKGIGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWSVIANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYKEG HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHKEEKNSNWLKYPSEYHVDFQVKRNRKTEILDQLPKNKISTAKVDSTFSYSSGGKFDSTKGIG RTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWSVIANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYKEG 2 the 2 LukA CC45 WT LukA CC45 WT ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHRNETNASWLKYPSEYHVDFQVQRNPKTEILDQLPKNKISTAKVDSTFSYSLGGKFDSTKGIGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWSVVANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYKEG ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHRNETNASWLKYPSEYHVDFQVQRNPKTEILDQLPKNKISTAKVDSTFSYSLGGKFDSTKGIGRTSS NSYSKSISYNQQNYDTIASGKNNNRHVHWSVVANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYKEG the 3 3 LukA CC8 W95E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile LukA CC8 W95 E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVK M QGSIHSNLKFESHKEEKNSNWLKYPSEYH I DFQVKRNRKTEILDQLPKNKISTAKVD A TFSYSSGGKFDSTKGIGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS I IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVK M QGSIHSNLKFESHKEEKNSNWLKYPSEYH I DFQVKRNRKTEILDQLPKNKISTAKVD A TFSYSSGGKFDSTKG IGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS I IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G 4 4 LukA CC45 W95E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile LukA CC45 W95 E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVK M QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKVD A TFSYSLGGKFDSTKGIGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVK M QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKVD A TFSYSLGGKFDSTKGIGRT SSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G 5 5 LukA CC8 W95W72 E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys LukA CC8 W95W72 E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPT C DKNVLLVK M QGSIHSNLKFESHKEEKNSNWLKYPSEYH I DFQVKRNRKTEILDQLPKNKISTAKV CA TFSYSSG C KFDSTK C IGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS I IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPT C DKNVLLVK M QGSIHSNLKFESHKEEKNSNWLKYPSEYH I DFQVKRNRKTEILDQLPKNKISTAKV CA TFSYSSG C KFDSTK C IGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS I IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G 6 6 LukA CC45 W95W72E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys LukA CC45 W95W72 E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPT C DKNVLLVK M QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKV CA TFSYSLG C KFDSTK C IGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPT C DKNVLLVK M QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKV CA TFSYSLG C KFDSTK C IG RTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G 7 7 LukA CC8 W97E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Thr246Val LukA CC8 W97 E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Thr246Val HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVK M QGSIHSNLKFESHKEEKNSNWLKYPSEYH I DFQVKRNRKTEILDQLPKNKISTAKVN A TFSYSSGGKFDSTKGIGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS I IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFL V YLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVK M QGSIHSNLKFESHKEEKNSNWLKYPSEYH I DFQVKRNRKTEILDQLPKNKISTAKVN A TFSYSSGGKFDSTKG IGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS I IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFL V YLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G 8 8 LukA CC45 W97 E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val LukA CC45 W97 E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVK M QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKVD A TFSYSLGGKFDSTKGIGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFL V YISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVK M QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKVD A TFSYSLGGKFDSTKGIGRT SSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFL V YISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G 9 9 LukA CC8 W97 W72E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Thr246Val, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys LukA CC8 W97 W72 E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Thr246Val, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPT C DKNVLLVK M QGSIHSNLKFESHKEEKNSNWLKYPSEYH I DFQVKRNRKTEILDQLPKNKISTAKV CA TFSYSSG C KFDSTK C IGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS I IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFL V YLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G HKDSQDQNKKEHVKSQKDKRNVTNVTNSTAPDDDDDIGKITKITKITKILQNLQFDFDFDFDFIDDPT C DKNVKFESNLKEKEKEKEKEKEKEKEKNSNWLKYPSEY QLPKISTAKISTAKISTAKISTAKISSSG C KFDSTK C IGRTSSNSYSKTISYDTIASGKNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNINNWHVHVHWS IINDLKNRINTRITVPELSkyryryryryryryryryryryryryryryryry work . SnektqfevtytrnqdilknrpgihyappileknkdgqrlivTyEVDWKNKTVDKYSDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDs 10 10 LukA CC45 W97 W72 E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys LukA CC45 W97 W72 E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPT C DKNVLLVK M QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKV CA TFSYSLG C KFDSTK C IGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFL V YISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPT C DKNVLLVK M QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKV CA TFSYSLG C KFDSTK C IG RTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFL V YISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G 11 11 LukA CC45 W94E321A, Lys81Leu, Ser139Ala, Val111Ile, Val191Ile LukA CC45 W94 E321A, Lys81Leu, Ser139Ala, Val111Ile, Val191Ile ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVK L QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKVD A TFSYSLGGKFDSTKGIGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVK L QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKVD A TFSYSLGGKFDSTKGIGRT SSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G 12 12 LukA CC45 W96E321A, Lys81Leu, Ser139Ala, Val111Ile, Val191Ile, Thr247Val LukA CC45 W96 E321A, Lys81Leu, Ser139Ala, Val111Ile, Val191Ile, Thr247Val ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVK L QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKVD A TFSYSLGGKFDSTKGIGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFL V YISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVK L QGSIHSNLKFESHRNETNASWLKYPSEYH I DFQVQRNPKTEILDQLPKNKISTAKVD A TFSYSLGGKFDSTKGIGRT SSNSYSKSISYNQQNYDTIASGKNNNRHVHWS I VANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFL V YISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G 13 13 LukA CC8Glu320Ala LukA CC8 Glu320Ala HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVK KQGSIHSNLKFESHKEEKNSNWLKYPSEYH VDFQVKRNRKTEILDQLPKNKISTAKVD STFSYSSGGKFDSTKGIGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS VIANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVK K QGSIHSNLKFESHKEEKNSNWLKYPSEYH V DFQVKRNRKTEILDQLPKNKISTAKVDS TFSYSSGGKFDSTKG IGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS V IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYK A G 14 14 LukA CC45Glu321Ala LukA CC45 Glu321Ala ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHRNETNASWLKYPSEYHVDFQVQRNPKTEILDQLPKNKISTAKVDSTFSYSLGGKFDSTKGIGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWSVVANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHRNETNASWLKYPSEYHVDFQVQRNPKTEILDQLPKNKISTAKVDSTFSYSLGGKFDSTKGIGRTSS NSYSKSISYNQQNYDTIASGKNNNRHVHWSVVANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKYSDQNKPYK A G 64 64 LukA CC8LukA CC8 ΔΔ 1010 HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVK KQGSIHSNLKFESHKEEKNSNWLKYPSEYH VDFQVKRNRKTEILDQLPKNKISTAKVD STFSYSSGGKFDSTKGIGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS VIANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKY HKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVK K QGSIHSNLKFESHKEEKNSNWLKYPSEYH V DFQVKRNRKTEILDQLPKNKISTAKVDS TFSYSSGGKFDSTKG IGRTSSNSYSKTISYNQQNYDTIASGKNNNWHVHWS V IANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKY 65 65 LukA CC45LukA CC45 ΔΔ 1010 ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHRNETNASWLKYPSEYHVDFQVQRNPKTEILDQLPKNKISTAKVDSTFSYSLGGKFDSTKGIGRTSSNSYSKSISYNQQNYDTIASGKNNNRHVHWSVVANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKY ANKDSQDQTKKEHVDKAQQKEKRNVNDKDKNTPGPDDIGKNGKVTKRTVSEYDKETNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHRNETNASWLKYPSEYHVDFQVQRNPKTEILDQLPKNKISTAKVDSTFSYSLGGKFDSTKGIGRTSS NSYSKSISYNQQNYDTIASGKNNNRHVHWSVVANDLKYGNEIKNRNDEFLFYRNTRLSTVENPELSFASKYRYPALVRSGFNPEFLTYISNEKTNDKTRFEVTYTRNQDILKNKPGIHYGQPILEQNKDGQRFIVVYEVDWKNKTVKVVEKY

金黃色葡萄球菌殺白細胞素 B(LukB) 變體 Staphylococcus aureus leukocidin B (LukB) variant

本發明的另一方面涉及金黃色葡萄球菌殺白細胞素B (LukB)變異多肽。這些LukB變異多肽包含一個或多個氨基酸殘基***、取代和/或缺失，它們提高了LukB的穩定性，從而有助於LukB類毒素的穩定性。如本文所述，這些LukB變異多肽是理想的疫苗抗原候選物，其可以單獨給藥或與殺白細胞素A (LukA)野生型或變異蛋白或多肽聯合給藥。當與LukA野生型或變異多肽聯合給藥時，所得類毒素模擬金黃色葡萄球菌LukAB毒素的結構，從而促進產生針對金黃色葡萄球菌最有效毒素之一的強大免疫應答。在任意的實施方案中，LukB變異多肽是全長LukB蛋白的變體，包含對應於全長成熟LukB蛋白序列的所有氨基酸殘基。在任意的實施方案中，LukB變體包含比全長成熟LukB蛋白更少的參考蛋白的氨基酸鏈。在一個實施方案中，LukB變異多肽長度至少為100個氨基酸殘基。在任意的實施方案中，LukB變異多肽是至少110、至少120、至少130、至少140、至少150、至少160、至少170、至少180、至少190、至少200、至少210、至少220、至少230、至少240、至少250、至少260、至少270、至少280、至少290、至少300個氨基酸殘基的長度。Another aspect of the invention relates to S. aureus leukocidin B (LukB) variant polypeptides. These LukB variant polypeptides contain one or more amino acid residue insertions, substitutions and/or deletions, which improve the stability of LukB, thereby contributing to the stability of LukB toxoids. As described herein, these LukB variant polypeptides are ideal vaccine antigen candidates that can be administered alone or in combination with leukocidin A (LukA) wild-type or variant proteins or polypeptides. When administered in combination with LukA wild-type or variant polypeptides, the resulting toxoid mimics the structure of the S. aureus LukAB toxin, thereby promoting a robust immune response against one of S. aureus' most potent toxins. In any embodiment, the LukB variant polypeptide is a variant of the full-length LukB protein, comprising all amino acid residues corresponding to the sequence of the full-length mature LukB protein. In any of the embodiments, the LukB variant comprises fewer amino acid chains of the reference protein than the full length mature LukB protein. In one embodiment, the LukB variant polypeptide is at least 100 amino acid residues in length. In any embodiment, the LukB variant polypeptide is at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, At least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300 amino acid residues in length.

雖然本文描述的示例性LukB變異多肽是克隆複合物CC8 (SEQ ID NO:15)和CC45 (SEQ ID NO:16)的LukB變體(見下表2)，但是本領域技術人員容易理解，鑒定LukB的氨基酸取代和/或缺失所基於的SEQ ID NO:15和SEQ ID NO:16是在各種克隆複合物之間保守的氨基酸殘基，或在各種克隆複合物之間高度保守的LukB區域內保守的氨基酸殘基。來自14個不同金黃色葡萄球菌菌株的LukB蛋白序列的比對(見圖2)顯示，本文中鑒定為易變異殘基的氨基酸殘基是在所有14個比對的LukB氨基酸序列中保守的殘基。雖然所鑒定的變異殘基的位置在各個LukB序列之間可能不同，但序列比對顯示了這些位置之間的對應關系。為了清楚起見，從序列比對中產生具有SEQ ID NO:39的氨基酸序列的LukB共有序列，並用於指定特定氨基酸變異的位置。例如，SEQ ID NO:39中穀氨酸殘基109位的氨基酸取代對應於SEQ ID NO:15、42、44和46–51的LukB序列中109位的穀氨酸殘基，對應於SEQ ID NO:41的LukB序列中110位的穀氨酸殘基，以及對應於SEQ ID NO:41的LukB序列中60位的穀氨酸殘基。因此，本文所述的經鑒定的氨基酸變體可以普遍應用於現在已知的或將來的任何LukB氨基酸序列中的相應氨基酸殘基。Although the exemplary LukB variant polypeptides described herein are LukB variants of cloning complexes CC8 (SEQ ID NO: 15) and CC45 (SEQ ID NO: 16) (see Table 2 below), those skilled in the art will readily appreciate that identifying Amino acid substitutions and/or deletions of LukB based on SEQ ID NO: 15 and SEQ ID NO: 16 are amino acid residues that are conserved among various cloning complexes, or within regions of LukB that are highly conserved among various cloning complexes Conserved amino acid residues. An alignment of LukB protein sequences from 14 different S. aureus strains (see Figure 2) revealed that the amino acid residues identified herein as easily variable residues were the ones conserved in all 14 aligned LukB amino acid sequences base. Although the positions of the identified variant residues may differ between individual LukB sequences, sequence alignments show correspondence between these positions. For clarity, a LukB consensus sequence having the amino acid sequence of SEQ ID NO: 39 was generated from the sequence alignment and used to designate the positions of specific amino acid variations. For example, the amino acid substitution at position 109 of glutamic acid residue in SEQ ID NO:39 corresponds to the glutamic acid residue at position 109 in the LukB sequence of SEQ ID NO:15, 42, 44 and 46-51, corresponding to SEQ ID NO:15, 42, 44 and 46-51. The glutamic acid residue at position 110 in the LukB sequence of NO:41, and the glutamic acid residue at position 60 in the LukB sequence corresponding to SEQ ID NO:41. Thus, the identified amino acid variants described herein can be generally applied to the corresponding amino acid residues in any now known or future LukB amino acid sequence.

在任意的實施方案中，本文公開的LukB變異多肽在對應於SEQ ID NO:39的氨基酸殘基Val53的氨基酸殘基上包含氨基酸取代或缺失。在任意的實施方案中，Val53處的氨基酸取代包括纈氨酸到亮氨酸(Val53Leu)的取代。在一個實施方案中，示例性的LukB變異多肽包含對應於SEQ ID NO:39中的Val53Leu取代的取代。In any embodiment, the LukB variant polypeptide disclosed herein comprises an amino acid substitution or deletion at an amino acid residue corresponding to amino acid residue Val53 of SEQ ID NO:39. In any of the embodiments, the amino acid substitution at Val53 comprises a valine to leucine (Val53Leu) substitution. In one embodiment, an exemplary LukB variant polypeptide comprises a substitution corresponding to the Val53Leu substitution in SEQ ID NO:39.

在任意的實施方案中，示例性的LukB變異多肽是CC8 LukB變異多肽，其在對應於SEQ ID NO:15的53位的氨基酸具有氨基酸取代。在任意的實施方案中，示例性的LukB變異多肽是CC8 LukB變異多肽，其在對應於SEQ ID NO:15的53位具有纈氨酸到亮氨酸的氨基酸取代。在任意的實施方案中，在53位具有纈氨酸到亮氨酸取代的示例性CC8 LukB序列包含SEQ ID NO:17的氨基酸序列，或具有與SEQ ID NO:17的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, an exemplary LukB variant polypeptide is a CC8 LukB variant polypeptide having an amino acid substitution at the amino acid corresponding to position 53 of SEQ ID NO:15. In any of the embodiments, an exemplary LukB variant polypeptide is a CC8 LukB variant polypeptide having a valine to leucine amino acid substitution at position 53 corresponding to SEQ ID NO:15. In any embodiment, an exemplary CC8 LukB sequence having a valine to leucine substitution at position 53 comprises, or is at least 85%, at least 85% identical to, the amino acid sequence of SEQ ID NO: 17. Amino acid sequences having at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity.

在任意的實施方案中，示例性的LukB變異多肽是CC45 LukB變異多肽，其在對應於SEQ ID NO:16的53位的氨基酸具有氨基酸取代。在任意的實施方案中，示例性的LukB變異多肽是CC45 LukB變異多肽，其在對應於SEQ ID NO:16的53位具有纈氨酸到亮氨酸的氨基酸取代。在一個實施方案中，包含對應於SEQ ID NO:39中的Val53Leu取代的示例性LukB變異多肽包含SEQ ID NO:18的氨基酸序列，或具有與SEQ ID NO:18的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, an exemplary LukB variant polypeptide is a CC45 LukB variant polypeptide having an amino acid substitution at the amino acid corresponding to position 53 of SEQ ID NO:16. In any of the embodiments, an exemplary LukB variant polypeptide is a CC45 LukB variant polypeptide having a valine to leucine amino acid substitution at position 53 corresponding to SEQ ID NO:16. In one embodiment, an exemplary LukB variant polypeptide comprising a substitution corresponding to Val53Leu in SEQ ID NO: 39 comprises the amino acid sequence of SEQ ID NO: 18, or has an amino acid sequence at least 85%, at least Amino acid sequences having 90%, at least 95%, at least 97% or at least 99% sequence similarity.

其他示例性的LukB變異多肽包括SEQ ID NO:40-51的任意一種LukB蛋白，所述LukB蛋白包含對應於Val53Leu的氨基酸取代。Other exemplary LukB variant polypeptides include any one of the LukB proteins of SEQ ID NO: 40-51 comprising an amino acid substitution corresponding to Val53Leu.

在任意的實施方案中，本文所述的LukB變異多肽包含對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的一個或多個氨基酸殘基上的氨基酸取代。在一個實施方案中，在一個或多個前述殘基上的氨基酸取代引入了能夠形成二硫鍵的半胱氨酸殘基，以穩定LukAB異二聚體結構的構象。例如，在一個實施方案中，本文所述的LukB變異蛋白或多肽對應於SEQ ID NO:39的Glu45 (Glu45Cys)的氨基酸殘基上包含穀氨酸到半胱氨酸的取代，並且在對應於SEQ ID NO:39的Thr121 (Thr121Cys)的氨基酸殘基上包含蘇氨酸到半胱氨酸的取代。45位和121位的這些半胱氨酸殘基形成二硫鍵，從而相對於野生型LukB或相對於本文所述的不含能形成二硫鍵的成對半胱氨酸殘基的其他變異LukB蛋白和多肽，增加了變異LukB的熱穩定性。In any of the embodiments, the LukB variant polypeptide described herein comprises an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39. In one embodiment, amino acid substitutions at one or more of the foregoing residues introduce cysteine residues capable of forming disulfide bonds to stabilize the conformation of the LukAB heterodimer structure. For example, in one embodiment, the LukB variant protein or polypeptide described herein comprises a substitution of glutamic acid to cysteine at an amino acid residue corresponding to Glu45 (Glu45Cys) of SEQ ID NO:39, and at an amino acid residue corresponding to Thr121 (Thr121Cys) of SEQ ID NO:39 comprises a threonine to cysteine substitution at the amino acid residue. These cysteine residues at positions 45 and 121 form a disulfide bond, thus relative to wild-type LukB or to other variations described herein that do not contain a pair of cysteine residues capable of disulfide bond formation LukB proteins and peptides that increase the thermostability of variant LukB.

在另一個實施方案中，本文所述的LukB變異蛋白或多肽在對應於SEQ ID NO:39的Glu109 (Glu109Cys)的氨基酸殘基上包含穀氨酸到半胱氨酸的取代，並且在對應於SEQ ID NO:39的Arg154 (Arg154Cys)的氨基酸殘基上包含精氨酸到半胱氨酸的取代。這些在109位和154位引入的半胱氨酸殘基形成二硫鍵，從而相對於野生型LukB或相對於不含能形成二硫鍵的成對半胱氨酸殘基的其他LukB變異多肽，增加了LukB變異體的熱穩定性。In another embodiment, the LukB variant protein or polypeptide described herein comprises a glutamic acid to cysteine substitution at an amino acid residue corresponding to Glu109 (Glu109Cys) of SEQ ID NO: 39, and at an amino acid residue corresponding to Arg154 (Arg154Cys) of SEQ ID NO: 39 comprises an arginine to cysteine substitution at the amino acid residue. These introduced cysteine residues at positions 109 and 154 form a disulfide bond, thereby disulphide-bonding relative to wild-type LukB or relative to other LukB variant polypeptides that do not contain a pair of cysteine residues capable of disulfide bond formation. , increased thermostability of LukB variants.

在任意的實施方案中，LukB變異多肽在對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，在任一氨基酸殘基上的氨基酸取代都包括如上所述的半胱氨酸殘基的引入。In any of the embodiments, the LukB variant polypeptide comprises amino acid substitutions at each of amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39. In any of the embodiments, amino acid substitutions at any amino acid residue include the introduction of a cysteine residue as described above.

在任意的實施方案中，LukB變異多肽是CC8 LukB變異多肽，其包含在對應於SEQ ID ID NO:15的氨基酸殘基Glu45、Glu109、Thr121和Arg154的任何一個或多個氨基酸殘基上的氨基酸取代。在任意的實施方案中，LukB變異多肽是CC8 LukB變異多肽，其在對應於SEQ ID NO:15的氨基酸殘基Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，任一氨基酸殘基上的氨基酸取代都包括如上所述的半胱氨酸殘基的引入。在一個實施方案中，包含在對應於SEQ ID ID NO:39的Glu45、Glu109、Thr121和Arg154的殘基處的半胱氨酸氨基酸取代的示例性LukB變異多肽包含SEQ ID NO:21的氨基酸序列，或具有與SEQ ID NO:21的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, the LukB variant polypeptide is a CC8 LukB variant polypeptide comprising an amino acid corresponding to any one or more of amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID ID NO: 15 replace. In any of the embodiments, the LukB variant polypeptide is a CC8 LukB variant polypeptide comprising an amino acid substitution at each of amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:15. In any of the embodiments, amino acid substitutions at any amino acid residue include the introduction of a cysteine residue as described above. In one embodiment, an exemplary LukB variant polypeptide comprising cysteine amino acid substitutions at residues corresponding to Glu45, Glu109, Thr121 and Arg154 of SEQ ID ID NO:39 comprises the amino acid sequence of SEQ ID NO:21 , or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:21.

在任意的實施方案中，LukB變異多肽是CC45 LukB變異多肽，其在對應於SEQ ID NO:16的氨基酸殘基Glu45、Glu110、Thr122和Arg155的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，LukB變異多肽是CC45 LukB變異多肽，其在對應於SEQ ID NO:16的氨基酸殘基Glu45、Glu110、Thr122和Arg155的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，任一氨基酸殘基上的氨基酸取代都包括如上所述的半胱氨酸殘基的引入。在另一個實施方案中，在對應於SEQ ID ID NO:39的Glu45、Glu110、Thr122和Arg155的殘基上包含半胱氨酸氨基酸取代的LukB變異多肽包含SEQ ID NO:22的氨基酸序列，或具有與SEQ ID NO:22的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any of the embodiments, the LukB variant polypeptide is a CC45 LukB variant polypeptide comprising an amino acid substitution at each of amino acid residues corresponding to amino acid residues Glu45, Glu110, Thr122 and Arg155 of SEQ ID NO:16. In any of the embodiments, the LukB variant polypeptide is a CC45 LukB variant polypeptide comprising an amino acid substitution at each of amino acid residues corresponding to amino acid residues Glu45, Glu110, Thr122 and Arg155 of SEQ ID NO:16. In any of the embodiments, amino acid substitutions at any amino acid residue include the introduction of a cysteine residue as described above. In another embodiment, the LukB variant polypeptide comprising cysteine amino acid substitutions at residues corresponding to Glu45, Glu110, Thr122 and Arg155 of SEQ ID ID NO:39 comprises the amino acid sequence of SEQ ID NO:22, or An amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:22.

其他示例性的LukB變異多肽包括SEQ ID NO:40-51的LukB蛋白中的任何一種，其在對應於SEQ ID NO:39的殘基Glu45、Glu109、Thr121和Arg154的殘基上包含所述氨基酸取代。Other exemplary LukB variant polypeptides include any of the LukB proteins of SEQ ID NO:40-51 comprising said amino acids at residues corresponding to residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39 replace.

在任意的實施方案中，本文公開的LukB變異多肽包含對應於SEQ ID NO:39的Val53的氨基酸殘基上的氨基酸取代，以及對應於SEQ ID NO:39的Glu45、Glu109、Thr121和Arg154的一個或多個氨基酸殘基上的氨基酸殘基取代。在任意的實施方案中，LukB變異多肽是CC8 LukB變異多肽，其在對應於SEQ ID NO:15的氨基酸殘基Val53、Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，LukB變異多肽是CC8 LukB變異多肽，其在對應於SEQ ID NO:15的氨基酸殘基Val53Leu、Glu45Cys、Glu109Cys、Thr121Cys和Arg154Cys的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，示例性的CC8 LukB變異多肽包含SEQ ID NO:19的氨基酸序列，或具有與SEQ ID NO:19的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any embodiment, the LukB variant polypeptide disclosed herein comprises an amino acid substitution corresponding to an amino acid residue of Val53 of SEQ ID NO:39, and one of Glu45, Glu109, Thr121 and Arg154 corresponding to SEQ ID NO:39 or amino acid residue substitutions on multiple amino acid residues. In any of the embodiments, the LukB variant polypeptide is a CC8 LukB variant polypeptide comprising an amino acid substitution at each of amino acid residues corresponding to amino acid residues Val53, Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:15. In any embodiment, the LukB variant polypeptide is a CC8 LukB variant polypeptide comprising an amino acid substitution at each of amino acid residues Val53Leu, Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys corresponding to SEQ ID NO:15. In any embodiment, the exemplary CC8 LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO: 19, or has at least 85%, at least 90%, at least 95%, at least 97% of the amino acid sequence of SEQ ID NO: 19 Or an amino acid sequence with at least 99% sequence similarity.

在任意的實施方案中，LukB變異多肽是CC45 LukB變異多肽，其在對應於SEQ ID NO:16的氨基酸殘基Val53、Glu45、Glu110、Thr122和Arg155的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，LukB變異多肽是CC45 LukB變異多肽，其在對應於SEQ ID NO:16的氨基酸殘基Val53Leu、Glu45Cys、Glu110Cys、Thr123Cys和Arg155Cys的每個氨基酸殘基上包含氨基酸取代。在對應於上述每個位置的殘基上具有氨基酸取代的示例性CC45 LukB變異多肽具有SEQ ID NO:20的氨基酸序列，或具有與SEQ ID NO:20的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In any of the embodiments, the LukB variant polypeptide is a CC45 LukB variant polypeptide comprising an amino acid substitution at each of amino acid residues corresponding to amino acid residues Val53, Glu45, Glu110, Thr122, and Arg155 of SEQ ID NO:16. In any embodiment, the LukB variant polypeptide is a CC45 LukB variant polypeptide comprising an amino acid substitution at each of amino acid residues Val53Leu, Glu45Cys, Glu110Cys, Thr123Cys and Arg155Cys corresponding to SEQ ID NO:16. Exemplary CC45 LukB variant polypeptides having amino acid substitutions at residues corresponding to each of the above positions have the amino acid sequence of SEQ ID NO: 20, or have an amino acid sequence at least 85%, at least 90%, Amino acid sequences of at least 95%, at least 97%, or at least 99% sequence similarity.

其他示例性的LukB變異多肽包括SEQ ID NO:40-51的LukB蛋白中的任一種，其在對應於SEQ ID NO:39的Val53、Glu45、Glu109、Thr121和Arg154的殘基上包含所述氨基酸取代。Other exemplary LukB variant polypeptides include any of the LukB proteins of SEQ ID NO:40-51 comprising said amino acids at residues corresponding to Val53, Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39 replace.

下表2提供了本文公開的示例性的變異LukB氨基酸序列。Table 2 below provides exemplary variant LukB amino acid sequences disclosed herein.

表surface 2.2. 示例性Exemplary LukBLuk B 多肽氨基酸序列Peptide Amino Acid Sequence SEQ ID NOSEQ ID NO 名稱name 描述describe 15 the 15 LukB CC8 WT the LukB CC8 WT KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLTEPNYDKETVFIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQDESREVKYTYGYKTGGDFSINRGGLTGNITKESNYSETISYQQPSYRTLLDQSTSHKGVGWKVEAHLINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLTEPNYDKETVFIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQDESREVKYTYGYKTGGDFSINRGGLTGNITKESNYSETISYQQPSYRTLLDQSTSHKGVGWKVEAHL INNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK the 16 the 16 LukB CC45 WT the LukB CC45 WT EIKSKITTVSEKNLDGDTKMYTRTATTSDTEKKISQSLQFNFLTEPNYDKETVFIKAKGTIGSGLKILNPNGYWNSTLRWPGSYSVSIQNVDDNNNSTNVTDFAPKNQDESREVKYTYGYKTGGDFSINRGGLTGNITKEKNYSETISYQQPSYRTLIDQPTTNKGVAWKVEAHSINNMGHDHTRQLTNDSDDRVKSEIFSLTRNGNLWAKDNFTPKNKMPVTVSEGFNPEFLAVMSHDKNDKGKSRFIVHYKRSMDDFKLDWNKHGFWGYWSGENHVDQKEEKLSALYEVDWKTHDVKLIKTFNDKEKK EIKSKITTVSEKNLDGDTKMYTRTATTSDTEKKISQSLQFNFLTEPNYDKETVFIKAKGTIGSGLKILNPNGYWNSTLRWPGSYSVSIQNVDDDNNNSTNVTDFAPKNQDESREVKYTYGYKTGGDFSINRGGLTGNITKEKNYSETISYQQPSYRTLIDQPTTNKGVAWKVEAHS INNMGHDHTRQLTNDSDDRVKSEIFSLTRNGNLWAKDNFTPKNKMPVTVSEGFNPEFLAVMSHDKNDKGKSRFIVHYKRSMDDFKLDWNKHGFWGYWSGENHVDQKEEKLSALYEVDWKTHDVKLIKTFNDKEKK 17 the 17 LukB CC8 Val53Leu the LukB CC8 Val53Leu KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLTEPNYDKET L FIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQDESREVKYTYGYKTGGDFSINRGGLTGNITKESNYSETISYQQPSYRTLLDQSTSHKGVGWKVEAHLINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLTEPNYDKET L FIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQDESREVKYTYGYKTGGDFSINRGGLTGNITKESNYSETISYQQPSYRTLLDQSTSHKGVGWKVEAH LINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK 18 the 18 LukB CC45 Val53Leu the LukB CC45 Val53Leu EIKSKITTVSEKNLDGDTKMYTRTATTSDTEKKISQSLQFNFLTEPNYDKET L FIKAKGTIGSGLKILNPNGYWNSTLRWPGSYSVSIQNVDDNNNSTNVTDFAPKNQDESREVKYTYGYKTGGDFSINRGGLTGNITKEKNYSETISYQQPSYRTLIDQPTTNKGVAWKVEAHSINNMGHDHTRQLTNDSDDRVKSEIFSLTRNGNLWAKDNFTPKNKMPVTVSEGFNPEFLAVMSHDKNDKGKSRFIVHYKRSMDDFKLDWNKHGFWGYWSGENHVDQKEEKLSALYEVDWKTHDVKLIKTFNDKEKK EIKSKITTVSEKNLDGDTKMYTRTATTSDTEKKISQSLQFNFLTEPNYDKET L FIKAKGTIGSGLKILNPNGYWNSTLRWPGSYSVSIQNVDDDNNNSTNVTDFAPKNQDESREVKYTYGYKTGGDFSINRGGLTGNITKEKNYSETISYQQPSYRTLIDQPTTNKGVAWKVEA HSINNMGHDHTRQLTNDSDDRVKSEIFSLTRNGNLWAKDNFTPKNKMPVTVSEGFNPEFLAVMSHDKNDKGKSRFIVHYKRSMDDFKLDWNKHGFWGYWSGENHVDQKEEKLSALYEVDWKTHDVKLIKTFNDKEKK 19 the 19 LukB CC8 Val53Leu, Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys the LukB CC8 Val53Leu, Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLT C PNYDKET L FIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQD C SREVKYTYGYK C GGDFSINRGGLTGNITKESNYSETISYQQPSY C TLLDQSTSHKGVGWKVEAHLINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLT C PNYDKET L FIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQD C SREVKYTYGYK C GGDFSINRGGLTGNITKESNYSETISYQQPSY C TLLDQSTSHKGVGW KVEAHLINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK 20 the 20 the LukB CC45 Val53Leu, Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys the LukB CC45 Val53Leu, Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys EIKSKITTVSEKNLDGDTKMYTRTATTSDTEKKISQSLQFNFLT C PNYDKET L FIKAKGTIGSGLKILNPNGYWNSTLRWPGSYSVSIQNVDDNNNSTNVTDFAPKNQD C SREVKYTYGYK C GGDFSINRGGLTGNITKEKNYSETISYQQPSY C TLIDQPTTNKGVAWKVEAHSINNMGHDHTRQLTNDSDDRVKSEIFSLTRNGNLWAKDNFTPKNKMPVTVSEGFNPEFLAVMSHDKNDKGKSRFIVHYKRSMDDFKLDWNKHGFWGYWSGENHVDQKEEKLSALYEVDWKTHDVKLIKTFNDKEKK EIKSKITTVSEKNLDGDTKMYTRTATTSDTEKKISQSLQFNFLT C PNYDKET L FIKAKGTIGSGLKILNPNGYWNSTLRWPGSYSVSIQNVDDDNNNSTNVTDFAPKNQD C SREVKYTYGYK C GGDFSINRGGLTGNITKEKNYSETISYQQPSY C TLIDQPTTNKGVA WKVEAHSINNMGHDHTRQLTNDSDDRVKSEIFSLTRNGNLWAKDNFTPKNKMPVTVSEGFNPEFLAVMSHDKNDKGKSRFIVHYKRSMDDFKLDWNKHGFWGYWSGENHVDQKEEKLSALYEVDWKTHDVKLIKTFNDKEKK 21 the twenty one LukB CC8 Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys the LukB CC8 Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLT C PNYDKET VFIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQD C SREVKYTYGYK C GGDFSINRGGLTGNITKESNYSETISYQQPSY C TLLDQSTSHKGVGWKVEAHLINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLT C PNYDKET V FIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQD C SREVKYTYGYK C GGDFSINRGGLTGNITKESNYSETISYQQPSY C TLLDQSTSHKGVGW KVEAHLINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK 22 the twenty two the LukB CC45 Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys the LukB CC45 Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys EIKSKITTVSEKNLDGDTKMYTRTATTSDTEKKISQSLQFNFLT C PNYDKET VFIKAKGTIGSGLKILNPNGYWNSTLRWPGSYSVSIQNVDDNNNSTNVTDFAPKNQD C SREVKYTYGYK C GGDFSINRGGLTGNITKEKNYSETISYQQPSY C TLIDQPTTNKGVAWKVEAHSINNMGHDHTRQLTNDSDDRVKSEIFSLTRNGNLWAKDNFTPKNKMPVTVSEGFNPEFLAVMSHDKNDKGKSRFIVHYKRSMDDFKLDWNKHGFWGYWSGENHVDQKEEKLSALYEVDWKTHDVKLIKTFNDKEKK EIKSKITTVSEKNLDGDTKMYTRTATTSDTEKKISQSLQFNFLT C PNYDKET V FIKAKGTIGSGLKILNPNGYWNSTLRWPGSYSVSIQNVDDNNNSTNVTDFAPKNQD C SREVKYTYGYK C GGDFSINRGGLTGNITKEKNYSETISYQQPSY C TLIDQPTTNKGVA WKVEAHSINNMGHDHTRQLTNDSDDRVKSEIFSLTRNGNLWAKDNFTPKNKMPVTVSEGFNPEFLAVMSHDKNDKGKSRFIVHYKRSMDDFKLDWNKHGFWGYWSGENHVDQKEEKLSALYEVDWKTHDVKLIKTFNDKEKK

根據本文公開內容的所有方面，本文公開的LukA和/或LukB變異多肽可以進一步包含一個或多個異源氨基酸序列。合適的異源氨基酸序列包括但不限於標簽序列、免疫原、信號序列等。合適的標簽序列包括但不限於聚組氨酸標簽、多聚精氨酸標簽、FLAG標簽、Step-標簽II、泛素標簽、NusA標簽、幾丁質結合域、鈣調蛋白結合肽、纖維素結合域、Hat標簽、S標簽、SBP、麥芽糖結合蛋白、穀胱甘肽S-轉移酶(參見Terpe K .，Terpe K., “Overview of Tag Protein Fusions: From Molecular and Biochemical Fundamentals to Commercial Systems,” Appl. Microbiol. Biotechnol. 60:523-33 (2003)，在此引入作為參考)。合適的免疫原包括但不限於T細胞表位、B細胞表位。合適的信號序列包括但不限於PelB信號序列、Sec信號序列、Tat信號序列、AmyE信號序列(參見Freudl R.，“Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems,” Microbial Cell Factories 17:52 (2018)，該文獻在此引入作為參考)。在任意的實施方案中，本文所述的LukA和/或LukB變異多肽包含PelB序列(MKYLLPTAAAGLLLLAAQPAMA；SEQ ID NO:23)。在任意的實施方案中，本文所述的LukA和/或LukB變異多肽包含His標簽(例如，NSAHHHHHHGS；SEQ ID NO:24)。在任意的實施方案中，本文所述的LukA和/或LukB變異多肽包含前述PelB序列和His標簽。According to all aspects of the disclosure herein, the LukA and/or LukB variant polypeptides disclosed herein may further comprise one or more heterologous amino acid sequences. Suitable heterologous amino acid sequences include, but are not limited to, tag sequences, immunogens, signal sequences, and the like. Suitable tag sequences include, but are not limited to, polyhistidine tags, polyarginine tags, FLAG tags, Step-tag II, ubiquitin tags, NusA tags, chitin binding domains, calmodulin binding peptides, cellulose Binding domain, Hat-tag, S-tag, SBP, maltose-binding protein, glutathione S-transferase (see Terpe K., Terpe K., “Overview of Tag Protein Fusions: From Molecular and Biochemical Fundamentals to Commercial Systems,” Appl. Microbiol. Biotechnol. 60:523-33 (2003), incorporated herein by reference). Suitable immunogens include, but are not limited to, T cell epitopes, B cell epitopes. Suitable signal sequences include, but are not limited to, the PelB signal sequence, the Sec signal sequence, the Tat signal sequence, the AmyE signal sequence (see Freudl R., "Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems," Microbial Cell Factories 17:52 (2018 ), which is hereby incorporated by reference). In any of the embodiments, the LukA and/or LukB variant polypeptides described herein comprise the PeIB sequence (MKYLLPTAAAGLLLLAAQPAMA; SEQ ID NO: 23). In any of the embodiments, the LukA and/or LukB variant polypeptides described herein comprise a His tag (eg, NSAHHHHHHGS; SEQ ID NO: 24). In any embodiment, the LukA and/or LukB variant polypeptides described herein comprise the aforementioned PelB sequence and a His tag.

變異Mutations LukALuka 和and LukBLuk B 多核苷酸和構建體polynucleotides and constructs

本發明的另一方面涉及編碼本文所述的LukA和LukB變異多肽的核酸分子。本文所述的核酸分子包括分離的多核苷酸、重組多核苷酸序列、部分表達載體或部分線性DNA或RNA序列，包括用於體外或體內轉錄/翻譯的線性DNA或RNA序列，以及與原核和真核細胞表達和分泌本文所述的LukA和LukB變異多肽兼容的載體。在任意的實施方案中，本文所述的LukA和LukB多核苷酸包含DNA。在任意的實施方案中，本文所述的LukA和LukB多核苷酸包含RNA，特別是mRNA。Another aspect of the invention pertains to nucleic acid molecules encoding the LukA and LukB variant polypeptides described herein. The nucleic acid molecules described herein include isolated polynucleotides, recombinant polynucleotide sequences, portions of expression vectors, or portions of linear DNA or RNA sequences, including linear DNA or RNA sequences for in vitro or in vivo transcription/translation, as well as those associated with prokaryotic and Eukaryotic cells express and secrete LukA and LukB variant polypeptide-compatible vectors described herein. In any of the embodiments, the LukA and LukB polynucleotides described herein comprise DNA. In any of the embodiments, the LukA and LukB polynucleotides described herein comprise RNA, particularly mRNA.

本發明的多核苷酸可以通過化學合成產生，例如在自動多核苷酸合成器上進行固相多核苷酸合成，並組裝成完整的單鏈或雙鏈分子。或者，本發明的多核苷酸可以通過其他技術產生，例如PCR，然後常規克隆。用於產生或獲得給定序列的多核苷酸的技術是本領域公知的。The polynucleotides of the present invention can be produced by chemical synthesis, such as solid-phase polynucleotide synthesis on an automated polynucleotide synthesizer, and assembled into complete single- or double-stranded molecules. Alternatively, polynucleotides of the invention can be produced by other techniques, such as PCR, followed by routine cloning. Techniques for generating or obtaining polynucleotides of a given sequence are well known in the art.

在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，該多肽在對應於SEQ ID NO:25的83位(Lys83Met)賴氨酸的殘基上包含賴氨酸到甲硫氨酸的取代。在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，其在對應於SEQ ID NO:25的141位 (Ser141Ala)的絲氨酸的殘基上包含絲氨酸到丙氨酸的取代。在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，該多肽在對應於SEQ ID NO:25的113位 (Val113Ile)的纈氨酸的殘基上包含纈氨酸到異亮氨酸的取代。在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，該多肽在對應於SEQ ID NO:25的193位 (Val193Ile)的纈氨酸的殘基上包含纈氨酸到異亮氨酸的取代。在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，其在對應於前述氨基酸殘基的殘基(即SEQ ID NO:25的Lys803Met、Ser141Ala、Val113Ile和Val193Ile)上包含賴氨酸到甲硫氨酸、絲氨酸到丙氨酸和纈氨酸到異亮氨酸的氨基酸取代。在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，其進一步包含對應於Glu323Ala的氨基酸取代，即多核苷酸編碼LukA變體，其包含對應於SEQ ID NO:25的Lys83Met、Ser141Ala、Val113Ile、Val193Ile和Glu323Ala取代的取代。In any embodiment, the polynucleotide of the present invention encodes a LukA variant polypeptide comprising lysine to methionine at a residue corresponding to lysine at position 83 (Lys83Met) of SEQ ID NO:25 of the replacement. In any embodiment, the polynucleotide of the invention encodes a LukA variant polypeptide comprising a serine to alanine substitution at the residue corresponding to serine at position 141 (Ser141Ala) of SEQ ID NO:25. In any embodiment, the polynucleotide of the present invention encodes a LukA variant polypeptide comprising valine to isoleucine at a residue corresponding to valine at position 113 (Val113Ile) of SEQ ID NO:25 acid substitution. In any embodiment, the polynucleotide of the present invention encodes a LukA variant polypeptide comprising valine to isoleucine at a residue corresponding to valine at position 193 (Val193Ile) of SEQ ID NO:25 acid substitution. In any embodiment, the polynucleotide of the present invention encodes a LukA variant polypeptide comprising lysine at residues corresponding to the aforementioned amino acid residues (i.e. Lys803Met, Ser141Ala, Val113Ile and Val193Ile of SEQ ID NO:25) Amino acid substitutions to methionine, serine to alanine, and valine to isoleucine. In any embodiment, the polynucleotide of the present invention encodes a LukA variant polypeptide, which further comprises an amino acid substitution corresponding to Glu323Ala, that is, the polynucleotide encodes a LukA variant, which comprises Lys83Met, Ser141Ala corresponding to SEQ ID NO:25 , Val113Ile, Val193Ile and Glu323Ala substitutions.

在一個實施方案中，示例性核酸分子是編碼CC8 LukA變體序列的核酸分子，例如編碼SEQ ID NO:1的變體，其包含對應於SEQ ID NO:1中Lys80Met、Ser138Ala、Val110Ile、Val190Ile和Glu320Ala的氨基酸取代。本文編碼CC8LukA的示例性核酸分子表述為SEQ ID NO:52。因此，在任意的實施方案中，示例性核酸分子是SEQ ID NO:52的變體，其中所述變體包含具有與SEQ ID NO:52的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。In one embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC8 LukA variant sequence, for example encoding a variant of SEQ ID NO: 1 comprising a sequence corresponding to Lys80Met, Ser138Ala, Val110Ile, Val190Ile and Amino acid substitution of Glu320Ala. An exemplary nucleic acid molecule encoding CC8LukA is represented herein as SEQ ID NO:52. Accordingly, in any embodiment, an exemplary nucleic acid molecule is a variant of SEQ ID NO:52, wherein the variant comprises a sequence having an amino acid sequence at least 85%, at least 90%, at least 95% identical to SEQ ID NO:52 , a nucleotide sequence of at least 97% or at least 99% sequence similarity.

在一個實施方案中，編碼CC8 Luk8變體的示例性核酸分子是編碼LukA變體序列(LukA CC8 Glu320Ala、Lys80Met、Ser138Ala、Val110Ile、Val190Ile)的核酸分子，所述LukA變體序列為SEQ ID NO:3或具有與SEQ ID NO:3的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。編碼這種LukA CC8變體的示例性核酸分子包含與SEQ ID NO:54具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。在任意的實施方案中，編碼這種LukACC8變異體的核酸分子包含SEQ ID NO:54的核苷酸序列。In one embodiment, an exemplary nucleic acid molecule encoding a CC8 Luk8 variant is a nucleic acid molecule encoding a LukA variant sequence (LukA CC8 Glu320Ala, Lys80Met, Ser138Ala, Val110Ile, Val190Ile), the LukA variant sequence being SEQ ID NO: 3 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:3. Exemplary nucleic acid molecules encoding such LukA CC8 variants comprise a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO:54. In any embodiment, the nucleic acid molecule encoding such LukACC8 variant comprises the nucleotide sequence of SEQ ID NO:54.

在另一個實施方案中，示例性核酸分子是編碼CC45 LukA變體序列的核酸分子，例如，編碼SEQ ID NO:2的變體，其包含對應於SEQ ID NO:2中Lys81Met、Ser139Ala、Val111Ile、Val191Ile和Glu321Ala的氨基酸取代。本文中編碼CC45 LukA的示例性核酸分子表述為SEQ ID NO:53。因此，在任意的實施方案中，示例性核酸分子是SEQ ID NO:53的變體，其中所述變體包含與SEQ ID NO:53的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。In another embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC45 LukA variant sequence, for example, encoding a variant of SEQ ID NO: 2 comprising a sequence corresponding to Lys81Met, Ser139Ala, Val111Ile, Amino acid substitutions of Val191Ile and Glu321Ala. An exemplary nucleic acid molecule encoding CC45 LukA is represented herein as SEQ ID NO:53. Accordingly, in any embodiment, an exemplary nucleic acid molecule is a variant of SEQ ID NO:53, wherein said variant comprises at least 85%, at least 90%, at least 95%, Nucleotide sequences with at least 97% or at least 99% sequence similarity.

在一個實施方案中，本發明的示例性核酸分子是編碼CC45 LukA變體序列(LukA CC45 Glu321Ala、Lys81Met、Ser139Ala、Val111Ile、Val191Ile)的核酸分子，所述CC45 LukA變體序列為SEQ ID NO:4或具有與SEQ ID NO:4的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。編碼這種LukA CC45變體的示例性核酸分子包含與SEQ ID NO:55具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。在任意的實施方案中，編碼這種LukACC8變異體的核酸分子包含SEQ ID NO:55的核苷酸序列。In one embodiment, an exemplary nucleic acid molecule of the present invention is a nucleic acid molecule encoding a CC45 LukA variant sequence (LukA CC45 Glu321Ala, Lys81Met, Ser139Ala, Val111Ile, Val191Ile), the CC45 LukA variant sequence being SEQ ID NO: 4 Or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:4. Exemplary nucleic acid molecules encoding such LukA CC45 variants comprise a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO:55. In any embodiment, the nucleic acid molecule encoding such LukACC8 variant comprises the nucleotide sequence of SEQ ID NO:55.

在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，所述多肽包含對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的一個或多個氨基酸殘基上的氨基酸取代。在一個實施方案中，所述多核苷酸編碼LukA變異多肽，該多肽在對應於SEQ ID NO:25的Tyr74 (Tyr74Cys)的氨基酸殘基上包含酪氨酸至半胱氨酸的取代，並且在對應於SEQ ID NO:25的Asp140 (Asp140Cys)的氨基酸殘基上包含天冬醯胺至半胱氨酸的取代。在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，所述多肽在對應於SEQ ID NO:25的Gly149 (Gly149Cys)的氨基酸殘基上包含甘氨酸到半胱氨酸的取代，並且對應於SEQ ID NO:25的Gly156 (Gly156Cys)的氨基酸殘基上包含甘氨酸到半胱氨酸的取代。在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，所述多肽在對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，這些氨基酸殘基中每一個的氨基酸取代都是如上所述的半胱氨酸殘基。In any embodiment, the polynucleotide of the present invention encodes a LukA variant polypeptide comprising one or more amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149 and Gly156 of SEQ ID NO:25 Amino acid substitutions. In one embodiment, the polynucleotide encodes a LukA variant polypeptide comprising a tyrosine to cysteine substitution at an amino acid residue corresponding to Tyr74 (Tyr74Cys) of SEQ ID NO: 25, and at The amino acid residue corresponding to Aspl40 (Asp140Cys) of SEQ ID NO: 25 comprises an asparagine to cysteine substitution. In any of the embodiments, the polynucleotide of the invention encodes a LukA variant polypeptide comprising a glycine to cysteine substitution at an amino acid residue corresponding to Gly149 (Gly149Cys) of SEQ ID NO: 25, and The amino acid residue corresponding to Gly156 (Gly156Cys) of SEQ ID NO: 25 comprises a glycine to cysteine substitution. In any embodiment, the polynucleotide of the invention encodes a LukA variant polypeptide comprising an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO:25 . In any of the embodiments, the amino acid substitution for each of these amino acid residues is a cysteine residue as described above.

在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，所述多肽包含在對應於Lys83、Ser141、Val113、Val193和Glu323的一個或多個氨基酸殘基上的氨基酸取代，以及對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的一個或多個氨基酸殘基上的氨基酸取代。在任意的實施方案中，所述多核苷酸編碼LukA變異多肽，所述多肽在對應於SEQ ID NO:25的殘基Lys83、Ser141、Val113、Val193和Glu323以及殘基Tyr74、Asp140、Gly149和Gly156的氨基酸殘基上包含氨基酸取代。In any embodiment, the polynucleotide of the present invention encodes a LukA variant polypeptide comprising an amino acid substitution at one or more amino acid residues corresponding to Lys83, Ser141, Val113, Val193, and Glu323, and corresponding to Amino acid substitutions at one or more of amino acid residues Tyr74, Aspl40, Gly149 and Gly156 of SEQ ID NO:25. In any of the embodiments, the polynucleotide encodes a LukA variant polypeptide at residues Lys83, Ser141, Val113, Val193 and Glu323 and residues Tyr74, Asp140, Gly149 and Gly156 corresponding to SEQ ID NO: 25 contains amino acid substitutions on the amino acid residues.

在一個實施方案中，示例性核酸分子是編碼CC8 LukA變體序列的核酸分子，例如，編碼SEQ ID NO:1的變體，其包含對應於SEQ ID NO:1的Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Glu320Ala、Tyr71Cys、Asp137Cys、Gly146Cys和Gly153Cys中每一個的氨基酸取代。在一個實施方案中，示例性核酸分子是編碼LukA變體序列(LukA CC8 Glu320Ala、Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Tyr71Cys、Asp137Cys、Gly146Cys、Gly153Cys)的核酸分子，所述LukA變體序列為SEQ ID NO:5或與SEQ ID NO:5的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In one embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC8 LukA variant sequence, for example, encoding a variant of SEQ ID NO:1 comprising Lys80Met, Ser138Ala, Val110Ile, Val190Ile corresponding to SEQ ID NO:1 Amino acid substitutions for each of , Glu320Ala, Tyr71Cys, Aspl37Cys, Gly146Cys and Gly153Cys. In one embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a LukA variant sequence (LukA CC8 Glu320Ala, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys), the LukA variant sequence being SEQ ID NO:5 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:5.

在另一個實施方案中，示例性核酸分子是編碼CC45 LukA變異體序列的核酸分子，例如，編碼SEQ ID NO:2的變體，其包含對應於SEQ ID NO:2的Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Glu321Ala、Tyr72Cys、Asp138Cys、Gly147Cys和Gly154Cys中每一個的氨基酸取代。在一個實施方案中，本發明的示例性核酸分子是編碼LukA變體序列(LukA CC45 Glu321Ala、Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Tyr72Cys、Asp138Cys、Gly147Cys、Gly154Cys)的核酸分子，所述LukA變體序列為SEQ ID NO:6或與該序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In another embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC45 LukA variant sequence, for example, encoding a variant of SEQ ID NO:2 comprising Lys81Met, Ser139Ala, Val111Ile, Amino acid substitutions for each of Val191Ile, Glu321Ala, Tyr72Cys, Aspl38Cys, Gly147Cys, and Gly154Cys. In one embodiment, an exemplary nucleic acid molecule of the present invention is a nucleic acid molecule encoding a LukA variant sequence (LukA CC45 Glu321Ala, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys), said LukA variant sequence is SEQ ID NO: 6 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to that sequence.

在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，該多肽在對應於SEQ ID NO:25的氨基酸殘基Thr249的氨基酸殘基處包含氨基酸的取代或缺失。在任意的實施方案中，多核苷酸編碼LukA變異體，該變異體在對應於SEQ ID NO:25的249位的殘基上包含蘇氨酸到纈氨酸的取代。在任意的實施方案中，本發明的多核苷酸編碼LukA變異多肽，該多肽包含在對應於Thr249的位置上的氨基酸取代，以及對應於SEQ ID NO:25的Lys83、Ser141、Val113、Val193、Glu323、Tyr74、Asp140、Gly149和Gly156的殘基上的任何一個或全部氨基酸取代。In any of the embodiments, the polynucleotide of the invention encodes a LukA variant polypeptide comprising an amino acid substitution or deletion at the amino acid residue corresponding to amino acid residue Thr249 of SEQ ID NO:25. In any of the embodiments, the polynucleotide encodes a LukA variant comprising a threonine to valine substitution at the residue corresponding to position 249 of SEQ ID NO:25. In any embodiment, the polynucleotide of the present invention encodes a LukA variant polypeptide comprising an amino acid substitution at a position corresponding to Thr249, and Lys83, Ser141, Val113, Val193, Glu323 corresponding to SEQ ID NO:25 Any one or all amino acid substitutions on residues of , Tyr74, Asp140, Gly149 and Gly156.

在一個實施方案中，示例性核酸分子是編碼CC8 LukA變體序列的核酸分子，例如，編碼SEQ ID NO:1的變體，其包含對應於SEQ ID NO:1的Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Glu320Ala和Thr246Val中每一個的氨基酸取代。在任意的實施方案中，本發明的示例性核酸分子是編碼LukA變體序列(LukA CC8 Glu320Ala、Lys80Met、Ser138Ala、Val110Ile、Val190Ile和Thr246Val)的核酸分子，所述LukA變體序列為SEQ ID NO:7或具有與SEQ ID NO:7的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。在任意的實施方案中，本發明的示例性核酸分子是編碼SEQ ID NO:9的CC8 LukA變體序列的核酸分子，其包含對應於Glu320Ala、Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Thr246Val、Tyr71Cys、Asp137Cys、Gly146Cys和Gly153Cys的氨基酸取代。本發明的示例性核酸分子包括編碼與SEQ ID NO:9的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列的核酸分子。在任意的實施方案中，編碼SEQ ID NO:9的這種LukA CC8變體的示例性核酸分子包含與SEQ ID NO:56具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。在任意的實施方案中，編碼這種LukACC8變體的核酸分子包含SEQ ID NO:56的核苷酸序列。In one embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC8 LukA variant sequence, for example, encoding a variant of SEQ ID NO:1 comprising Lys80Met, Ser138Ala, Val110Ile, Val190Ile corresponding to SEQ ID NO:1 Amino acid substitutions for each of , Glu320Ala, and Thr246Val. In any embodiment, the exemplary nucleic acid molecule of the present invention is a nucleic acid molecule encoding a LukA variant sequence (LukA CC8 Glu320Ala, Lys80Met, Ser138Ala, Val110Ile, Val190Ile and Thr246Val), said LukA variant sequence being SEQ ID NO: 7 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:7. In any embodiment, the exemplary nucleic acid molecule of the present invention is a nucleic acid molecule encoding the CC8 LukA variant sequence of SEQ ID NO: 9, which comprises a sequence corresponding to Glu320Ala, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Thr246Val, Tyr71Cys, Asp137Cys , Gly146Cys and Gly153Cys amino acid substitutions. Exemplary nucleic acid molecules of the invention include nucleic acid molecules that encode an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similar to the amino acid sequence of SEQ ID NO:9. In any embodiment, an exemplary nucleic acid molecule encoding such a LukA CC8 variant of SEQ ID NO:9 comprises at least 85%, at least 90%, at least 95%, at least 97%, or at least Nucleotide sequences with 99% sequence similarity. In any embodiment, the nucleic acid molecule encoding such LukACC8 variant comprises the nucleotide sequence of SEQ ID NO:56.

在另一個實施方案中，示例性核酸分子是編碼CC45 LukA變體序列的核酸分子，例如，編碼SEQ ID NO:2的變體，其包含對應於SEQ ID NO:2的Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Glu321Ala和Thr247Val中每一個的氨基酸取代。在任意的實施方案中，本發明的示例性核酸分子是編碼LukA變體序列(LukA CC45 Glu321Ala、Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Thr247Val)的核酸分子，所述LukA變體序列為SEQ ID NO:8或具有與SEQ ID NO:8的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。在任意的實施方案中，本發明的示例性核酸分子是編碼SEQ ID NO:10的LukA變體序列的核酸分子，其包含對應於Glu321Ala、Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Thr247Val、Tyr72Cys、Asp138Cys、Gly147Cys和Gly154Cys的氨基酸取代。本發明的示例性核酸分子包括編碼與SEQ ID NO:10的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列的核酸分子。在任意的實施方案中，編碼SEQ ID NO:10的這種LukA CC45變體的示例性核酸分子包含與SEQ ID NO:57具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。在任意的實施方案中，編碼這種LukACC8變體的核酸分子包含SEQ ID NO:57的核苷酸序列。In another embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC45 LukA variant sequence, for example, encoding a variant of SEQ ID NO:2 comprising Lys81Met, Ser139Ala, Val111Ile, Amino acid substitutions for each of Val191Ile, Glu321Ala, and Thr247Val. In any embodiment, the exemplary nucleic acid molecule of the present invention is a nucleic acid molecule encoding a LukA variant sequence (LukA CC45 Glu321Ala, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val), said LukA variant sequence being SEQ ID NO: 8 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:8. In any embodiment, the exemplary nucleic acid molecule of the present invention is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO: 10, which comprises a sequence corresponding to Glu321Ala, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val, Tyr72Cys, Asp138Cys, Amino acid substitutions of Gly147Cys and Gly154Cys. Exemplary nucleic acid molecules of the invention include nucleic acid molecules that encode an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similar to the amino acid sequence of SEQ ID NO: 10. In any embodiment, an exemplary nucleic acid molecule encoding such a LukA CC45 variant of SEQ ID NO: 10 comprises at least 85%, at least 90%, at least 95%, at least 97%, or at least Nucleotide sequences with 99% sequence similarity. In any embodiment, the nucleic acid molecule encoding such a LukACC8 variant comprises the nucleotide sequence of SEQ ID NO:57.

本發明的另一方面涉及編碼本文公開的LukB變異多肽的多核苷酸。在一個實施方案中，多核苷酸編碼LukB變異多肽，該多肽在對應於SEQ ID NO:39的氨基酸殘基Val53的氨基酸殘基上包含氨基酸取代或缺失。在任意的實施方案中，Val53處的氨基酸取代包括纈氨酸到亮氨酸(Val53Leu)的取代。在一個實施方案中，示例性核酸分子是編碼CC8 LukB變體序列的核酸分子，例如，編碼SEQ ID NO:15的變體，其包含SEQ ID NO:15的53位處的氨基酸取代。編碼CC8 LukB的示例性核酸分子在此表述為SEQ ID NO:58。因此，在任意的實施方案中，示例性核酸分子是SEQ ID NO:58的變體，其中所述變體包含與SEQ ID NO:58的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。Another aspect of the invention pertains to polynucleotides encoding the LukB variant polypeptides disclosed herein. In one embodiment, the polynucleotide encodes a LukB variant polypeptide comprising an amino acid substitution or deletion at an amino acid residue corresponding to amino acid residue Val53 of SEQ ID NO:39. In any of the embodiments, the amino acid substitution at Val53 comprises a valine to leucine (Val53Leu) substitution. In one embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC8 LukB variant sequence, eg, encoding a variant of SEQ ID NO:15 comprising an amino acid substitution at position 53 of SEQ ID NO:15. An exemplary nucleic acid molecule encoding CC8 LukB is represented herein as SEQ ID NO:58. Accordingly, in any embodiment, the exemplary nucleic acid molecule is a variant of SEQ ID NO:58, wherein the variant comprises at least 85%, at least 90%, at least 95%, Nucleotide sequences with at least 97% or at least 99% sequence similarity.

在一個實施方案中，本發明的示例性核酸分子編碼LukB變異多肽，所述變異多肽為SEQ ID NO:17 (LukB CC8 V53L)或具有與SEQ ID NO:17的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。編碼這種LukB CC8 V53L變體的示例性核酸分子包含具有與SEQ ID NO:60至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。在任意的實施方案中，編碼這種LukACC8變體的核酸分子包含SEQ ID NO:60的核苷酸序列。In one embodiment, the exemplary nucleic acid molecule of the present invention encodes a LukB variant polypeptide that is SEQ ID NO: 17 (LukB CC8 V53L) or has an amino acid sequence that is at least 85%, at least 90% identical to SEQ ID NO: 17 %, at least 95%, at least 97%, or at least 99% sequence similarity to amino acid sequences. Exemplary nucleic acid molecules encoding such LukB CC8 V53L variants comprise a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO:60. In any embodiment, the nucleic acid molecule encoding such LukACC8 variant comprises the nucleotide sequence of SEQ ID NO:60.

在另一個實施方案中，示例性核酸分子是編碼CC45 LukB變體序列的核酸分子，例如，編碼SEQ ID NO:16的變體，其包含SEQ ID NO:16的53位的氨基酸取代。編碼CC45 LukB的示例性核酸分子在此表述為SEQ ID NO:59。因此，在任意的實施方案中，示例性核酸分子是SEQ ID NO:59的變體，其中所述變體包含與SEQ ID NO:59的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。In another embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC45 LukB variant sequence, eg, encoding a variant of SEQ ID NO:16 comprising an amino acid substitution at position 53 of SEQ ID NO:16. An exemplary nucleic acid molecule encoding CC45 LukB is represented herein as SEQ ID NO:59. Therefore, in any embodiment, the exemplary nucleic acid molecule is a variant of SEQ ID NO: 59, wherein the variant comprises at least 85%, at least 90%, at least 95%, Nucleotide sequences with at least 97% or at least 99% sequence similarity.

在另一個實施方案中，本發明的示例性多核苷酸編碼LukB變異多肽，所述變異多肽為SEQ ID NO:18 (LukB CC45 V53L)或具有與SEQ ID NO:18的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。編碼這種LukB CC45 V53L變體的示例性核酸分子包含具有與SEQ ID NO:61至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列。在任意的實施方案中，編碼這種LukACC45變體的核酸分子包含SEQ ID NO:61的核苷酸序列。In another embodiment, an exemplary polynucleotide of the invention encodes a LukB variant polypeptide that is SEQ ID NO: 18 (LukB CC45 V53L) or has an amino acid sequence that is at least 85% identical to SEQ ID NO: 18, Amino acid sequences having at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity. Exemplary nucleic acid molecules encoding such LukB CC45 V53L variants comprise a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO:61. In any embodiment, the nucleic acid molecule encoding such LukACC45 variant comprises the nucleotide sequence of SEQ ID NO:61.

在任意的實施方案中，本發明的多核苷酸編碼LukB變異多肽，該多肽包含對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的一個或多個氨基酸殘基上的氨基酸取代。在一個實施方案中，在一個或多個前述殘基上的氨基酸取代引入了一個或多個能夠形成二硫鍵的半胱氨酸殘基，以穩定LukAB異二聚體結構的構象。在一個實施方案中，所述多核苷酸編碼LukB變異蛋白或多肽，所述蛋白或多肽包含對應於SEQ ID NO:39的Glu45 (Glu45Cys)的氨基酸殘基上的穀氨酸到半胱氨酸的取代，以及對應於SEQ ID NO:39的Thr121 (Thr121Cys)的氨基酸殘基上的蘇氨酸到半胱氨酸的取代。在另一個實施方案中，多核苷酸編碼LukB變異蛋白或多肽，其包含對應於SEQ ID NO:39的Glu109 (Glu109Cys)的氨基酸殘基上的穀氨酸到半胱氨酸的取代，以及對應於SEQ ID NO:39的Arg154 (Arg154Cys)的氨基酸殘基上的精氨酸到半胱氨酸的取代。In any embodiment, the polynucleotide of the present invention encodes a LukB variant polypeptide comprising amino acids corresponding to one or more of amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39 replace. In one embodiment, amino acid substitutions at one or more of the foregoing residues introduce one or more cysteine residues capable of disulfide bond formation to stabilize the conformation of the LukAB heterodimer structure. In one embodiment, the polynucleotide encodes a LukB variant protein or polypeptide comprising glutamic acid to cysteine at the amino acid residue corresponding to Glu45 (Glu45Cys) of SEQ ID NO:39 and a substitution of threonine to cysteine at the amino acid residue corresponding to Thr121 (Thr121Cys) of SEQ ID NO:39. In another embodiment, the polynucleotide encodes a LukB variant protein or polypeptide comprising a glutamic acid to cysteine substitution at an amino acid residue corresponding to Glu109 (Glu109Cys) of SEQ ID NO: 39, and a substitution corresponding to Arginine to cysteine substitution at amino acid residue Arg154 (Arg154Cys) of SEQ ID NO:39.

在任意的實施方案中，本發明的多核苷酸編碼LukB變異多肽，該多肽在對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，任一氨基酸殘基上的氨基酸取代都包括如上所述的半胱氨酸殘基的引入。In any of the embodiments, the polynucleotide of the invention encodes a LukB variant polypeptide comprising an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39. In any of the embodiments, amino acid substitutions at any amino acid residue include the introduction of a cysteine residue as described above.

在一個實施方案中，示例性核酸分子是編碼CC8 LukB變體序列的核酸分子，例如，編碼SEQ ID NO:15的變體，所述變體在對應於SEQ ID NO:15的氨基酸殘基Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上包含氨基酸取代。在一個實施方案中，多核苷酸編碼LukB變異多肽，其包含SEQ ID NO:21的氨基酸序列(LukB CC8 Glu45Cys、Glu109Cys、Thr121Cys和Arg154Cys)或具有與SEQ ID NO:21的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。在另一個實施方案中，示例性核酸分子是編碼CC45 LukB變體序列的核酸分子，例如編碼SEQ ID NO:16的變體，其在對應於SEQ ID NO:16的氨基酸殘基Glu45、Glu110、Thr122和Arg155的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，多核苷酸編碼LukB變異多肽，該多肽包含SEQ ID NO:22的氨基酸序列(LukB CC45 Glu45Cys、Thr122Cys、Glu110Cys、Arg155Cys)或具有與SEQ ID NO:22的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列。In one embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC8 LukB variant sequence, for example, encoding a variant of SEQ ID NO: 15 at amino acid residue Glu45 corresponding to SEQ ID NO: 15 , Glu109, Thr121 and Arg154 contain amino acid substitutions at each amino acid residue. In one embodiment, the polynucleotide encodes a LukB variant polypeptide comprising the amino acid sequence of SEQ ID NO: 21 (LukB CC8 Glu45Cys, Glu109Cys, Thr121Cys and Arg154Cys) or having at least 85% of the amino acid sequence of SEQ ID NO: 21, Amino acid sequences having at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity. In another embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC45 LukB variant sequence, for example encoding a variant of SEQ ID NO: 16 at amino acid residues Glu45, Glu110, Glu110, Amino acid substitutions are included at each amino acid residue of Thr122 and Arg155. In any embodiment, the polynucleotide encodes a LukB variant polypeptide comprising the amino acid sequence of SEQ ID NO: 22 (LukB CC45 Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys) or having an amino acid sequence at least 85 from the amino acid sequence of SEQ ID NO: 22 %, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to amino acid sequences.

在任意的實施方案中，本發明的多核苷酸編碼LukB變異多肽，其包含對應於SEQ ID NO:39的Val53的氨基酸殘基上的氨基酸取代，以及對應於SEQ ID NO:39的Glu45、Glu109、Thr121和Arg154的一個或多個氨基酸殘基上的氨基酸殘基取代。在一個實施方案中，示例性核酸分子是編碼CC8 LukB變體序列的核酸分子，例如，編碼SEQ ID NO:15的變體，其在對應於SEQ ID NO:15的氨基酸殘基Val53、Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，多核苷酸編碼具有SEQ ID NO:19的氨基酸序列(LukB CC8 Val53Leu、Glu45Cys、Glu109Cys、Thr121Cys和Arg154Cys)的LukB變異多肽，或者編碼具有與SEQ ID NO:19的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列的LukB變異多肽。在另一個實施方案中，示例性核酸分子是編碼CC45 LukB變體序列的核酸分子，例如，編碼SEQ ID NO:16的變體，其在對應於SEQ ID NO:16的氨基酸殘基Val53、Glu45、Glu110、Thr122和Arg155的每個氨基酸殘基上包含氨基酸取代。在任意的實施方案中，多核苷酸編碼具有SEQ ID NO:20的氨基酸序列(LukB CC45 Val53Leu、Glu45Cys、Thr122Cys、Glu110Cys、Arg155Cys)的LukB變異多肽，或編碼具有與SEQ ID NO:20的氨基酸序列至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的氨基酸序列的LukB變異多肽。In any embodiment, the polynucleotide of the present invention encodes a LukB variant polypeptide comprising an amino acid substitution corresponding to the amino acid residue of Val53 of SEQ ID NO:39, and corresponding to Glu45, Glu109 of SEQ ID NO:39 Amino acid residue substitutions on one or more amino acid residues of , Thr121 and Arg154. In one embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC8 LukB variant sequence, for example, encoding a variant of SEQ ID NO: 15 at amino acid residues Val53, Glu45, Amino acid substitutions were included at each amino acid residue of Glu109, Thr121 and Arg154. In any embodiment, the polynucleotide encodes a LukB variant polypeptide having the amino acid sequence of SEQ ID NO: 19 (LukB CC8 Val53Leu, Glu45Cys, Glu109Cys, Thr121Cys and Arg154Cys), or encodes a LukB variant polypeptide having the amino acid sequence of SEQ ID NO: 19 A LukB variant polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity. In another embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC45 LukB variant sequence, e.g., encoding a variant of SEQ ID NO: 16 at amino acid residues Val53, Glu45 corresponding to SEQ ID NO: 16 , Glu110, Thr122 and Arg155 contain amino acid substitutions at each amino acid residue. In any embodiment, the polynucleotide encodes a LukB variant polypeptide having the amino acid sequence of SEQ ID NO:20 (LukB CC45 Val53Leu, Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys), or encodes a LukB variant polypeptide having the amino acid sequence of SEQ ID NO:20 A LukB variant polypeptide having an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity.

在另一個實施方案中，本發明的示例性核酸分子是編碼本文公開的LukA序列和LukB序列的核酸分子。在一個實施方案中，示例性核酸分子是編碼CC45 LukA序列(變體或非變體)和CC45 LukB序列(變體或非變體)的多核苷酸。例如，編碼本文公開的CC45 LukA變體序列和CC45 LukB非變體序列的多核苷酸，或編碼本文公開的CC45 LukA非變體序列和CC45 LukB變體序列的多核苷酸。In another embodiment, an exemplary nucleic acid molecule of the invention is a nucleic acid molecule encoding a LukA sequence and a LukB sequence disclosed herein. In one embodiment, an exemplary nucleic acid molecule is a polynucleotide encoding a CC45 LukA sequence (variant or non-variant) and a CC45 LukB sequence (variant or non-variant). For example, a polynucleotide encoding a CC45 LukA variant sequence and a CC45 LukB non-variant sequence disclosed herein, or a polynucleotide encoding a CC45 LukA non-variant sequence and a CC45 LukB variant sequence disclosed herein.

在另一個實施方案中，示例性核酸分子是編碼CC8 LukA序列(變體或非變體)和CC8 LukB序列(變體或非變體)的多核苷酸。例如，編碼本文公開的CC8 LukA變體序列和CC8 LukB非變體序列的多核苷酸，或編碼本文公開的CC8 LukA非變體序列和CC8 LukB變體序列的多核苷酸。In another embodiment, an exemplary nucleic acid molecule is a polynucleotide encoding a CC8 LukA sequence (variant or non-variant) and a CC8 LukB sequence (variant or non-variant). For example, a polynucleotide encoding a CC8 LukA variant sequence and a CC8 LukB non-variant sequence disclosed herein, or a polynucleotide encoding a CC8 LukA non-variant sequence and a CC8 LukB variant sequence disclosed herein.

在另一個實施方案中，示例性核酸分子是編碼CC45 LukA序列(變體或非變體)和CC8 LukB序列(變體或非變體)的多核苷酸。例如，編碼本文公開的CC45 LuCA變體序列和CC8 LukB非變體序列的多核苷酸，或編碼本文公開的CC45 LuCA非變體序列和CC8 LukB變體序列的多核苷酸。In another embodiment, an exemplary nucleic acid molecule is a polynucleotide encoding a CC45 LukA sequence (variant or non-variant) and a CC8 LukB sequence (variant or non-variant). For example, a polynucleotide encoding a CC45 LuCA variant sequence and a CC8 LukB non-variant sequence disclosed herein, or a polynucleotide encoding a CC45 LuCA non-variant sequence and a CC8 LukB variant sequence disclosed herein.

在另一個實施方案中，示例性核酸分子是編碼CC8 LukA序列(變體或非變體)和CC45 LukB序列(變體或非變體)的核酸分子。例如，編碼本文公開的CC8 LukA變體序列和CC45 LukB非變體序列的多核苷酸，或編碼本文公開的CC8 LukA非變體序列和CC45 LukB變體序列的多核苷酸。In another embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC8 LukA sequence (variant or non-variant) and a CC45 LukB sequence (variant or non-variant). For example, a polynucleotide encoding a CC8 LukA variant sequence and a CC45 LukB non-variant sequence disclosed herein, or a polynucleotide encoding a CC8 LukA non-variant sequence and a CC45 LukB variant sequence disclosed herein.

在另一個實施方案中，本發明的示例性核酸分子是編碼LukA變體序列和LukB野生型序列的多核苷酸。例如，編碼選自SEQ ID NO:3、4、5、6、7、8、9、10、11、12、13或14中任一項所述的LukA變體序列的多核苷酸，與SEQ ID NO:15或SEQ ID NO:16的LukB野生型(即非變體)序列組合。In another embodiment, an exemplary nucleic acid molecule of the invention is a polynucleotide encoding a LukA variant sequence and a LukB wild-type sequence. For example, a polynucleotide encoding a LukA variant sequence selected from any one of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and SEQ ID NO: Combinations of LukB wild-type (ie non-variant) sequences of ID NO: 15 or SEQ ID NO: 16.

在任意的實施方案中，本發明的示例性核酸分子是編碼LukA野生型序列和LukB變體序列的多核苷酸。例如，編碼SEQ ID NO:1或SEQ ID NO:2的LukA野生型序列與選自SEQ ID NO:17、18、19、20、21或22中任一個的LukB變體序列組合的多核苷酸。In any of the embodiments, an exemplary nucleic acid molecule of the invention is a polynucleotide encoding a LukA wild-type sequence and a LukB variant sequence. For example, a polynucleotide encoding a LukA wild-type sequence of SEQ ID NO: 1 or SEQ ID NO: 2 combined with a LukB variant sequence selected from any one of SEQ ID NO: 17, 18, 19, 20, 21 or 22 .

在任意的實施方案中，本發明的示例性核酸分子是編碼LukA變體序列和LukB變體序列的多核苷酸。例如，編碼選自SEQ ID NO:3、4、5、6、7、8、9、10、11、12、13或14中任一個的LukA變體序列的多核苷酸，與選自SEQ ID NO:17、18、19、20、21或22中任一個的LukB變體序列組合。In any of the embodiments, an exemplary nucleic acid molecule of the invention is a polynucleotide encoding a LukA variant sequence and a LukB variant sequence. For example, a polynucleotide encoding a LukA variant sequence selected from any one of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and selected from SEQ ID A combination of LukB variant sequences of any one of NO: 17, 18, 19, 20, 21 or 22.

在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:3的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列、SEQ ID NO:17或18的LukB變體序列、SEQ ID NO:19或20的LukB變體序列或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:4的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列、SEQ ID NO:17或18的LukB變體序列、SEQ ID NO:19或20的LukB變體序列或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:5的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列、SEQ ID NO:17或18的LukB變體序列、SEQ ID NO:19或20的LukB變體序列或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:6的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列、SEQ ID NO:17或18的LukB變體序列、SEQ ID NO:19或20的LukB變體序列或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:7的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列、SEQ ID NO:17或18的LukB變體序列、SEQ ID NO:19或20的LukB變體序列或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:8的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列、SEQ ID NO:17或18的LukB變體序列、SEQ ID NO:19或20的LukB變體序列或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:9的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列、SEQ ID NO:17或18的LukB變體序列、SEQ ID NO:19或20的LukB變體序列或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:10的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列，SEQ ID NO:17或18的LukB變體序列，SEQ ID NO:19或20的LukB變體序列，或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:11的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列，SEQ ID NO:17或18的LukB變體序列，SEQ ID NO:19或20的LukB變體序列，或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:12的LukA變體序列和SEQ ID NO:15或16的LukB非變體序列，SEQ ID NO:17或18的LukB變體序列，SEQ ID NO:19或20的LukB變體序列，或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:13的LukA變體序列和SEQ ID NO:17或18的LukB變體序列，SEQ ID NO:19或20的LukB變體序列，或SEQ ID NO:21或22的LukB變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:14的LukA變體序列和SEQ ID NO:17或18的LukB變體序列，SEQ ID NO:19或20的LukB變體序列，或SEQ ID NO:21或22的LukB變體序列。In any embodiment, the exemplary nucleic acid molecule of the invention encodes a LukA variant sequence of SEQ ID NO:3 and a LukB non-variant sequence of SEQ ID NO:15 or 16, a LukB variant of SEQ ID NO:17 or 18 sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the invention encodes a LukA variant sequence of SEQ ID NO:4 and a LukB non-variant sequence of SEQ ID NO:15 or 16, a LukB variant of SEQ ID NO:17 or 18 sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the invention encodes a LukA variant sequence of SEQ ID NO:5 and a LukB non-variant sequence of SEQ ID NO:15 or 16, a LukB variant of SEQ ID NO:17 or 18 sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the invention encodes a LukA variant sequence of SEQ ID NO:6 and a LukB non-variant sequence of SEQ ID NO:15 or 16, a LukB variant of SEQ ID NO:17 or 18 sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the invention encodes a LukA variant sequence of SEQ ID NO:7 and a LukB non-variant sequence of SEQ ID NO:15 or 16, a LukB variant of SEQ ID NO:17 or 18 sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the invention encodes a LukA variant sequence of SEQ ID NO:8 and a LukB non-variant sequence of SEQ ID NO:15 or 16, a LukB variant of SEQ ID NO:17 or 18 sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the invention encodes a LukA variant sequence of SEQ ID NO:9 and a LukB non-variant sequence of SEQ ID NO:15 or 16, a LukB variant of SEQ ID NO:17 or 18 sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the present invention encodes the LukA variant sequence of SEQ ID NO:10 and the LukB non-variant sequence of SEQ ID NO:15 or 16, the LukB variant of SEQ ID NO:17 or 18 A variant sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the present invention encodes the LukA variant sequence of SEQ ID NO:11 and the LukB non-variant sequence of SEQ ID NO:15 or 16, the LukB variant of SEQ ID NO:17 or 18 A variant sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecule of the present invention encodes the LukA variant sequence of SEQ ID NO:12 and the LukB non-variant sequence of SEQ ID NO:15 or 16, the LukB variant of SEQ ID NO:17 or 18 A variant sequence, a LukB variant sequence of SEQ ID NO: 19 or 20, or a LukB variant sequence of SEQ ID NO: 21 or 22. In any embodiment, the exemplary nucleic acid molecules of the present invention encode the LukA variant sequence of SEQ ID NO:13 and the LukB variant sequence of SEQ ID NO:17 or 18, the LukB variant of SEQ ID NO:19 or 20 sequence, or the LukB variant sequence of SEQ ID NO:21 or 22. In any embodiment, the exemplary nucleic acid molecules of the present invention encode the LukA variant sequence of SEQ ID NO:14 and the LukB variant sequence of SEQ ID NO:17 or 18, the LukB variant of SEQ ID NO:19 or 20 sequence, or the LukB variant sequence of SEQ ID NO:21 or 22.

在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:17的LukB變體序列和SEQ ID NO:1或2的LukA非變體序列，SEQ ID NO:3或4的LukA變體序列，SEQ ID NO:5或6的LukA變體序列，SEQ ID NO:7或8的LukA變體序列，SEQ ID NO:9或10的LukA變體序列，SEQ ID NO:11或12的LukA變體序列，或SEQ ID NO:13或14的LukA變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:18的LukB變體序列和SEQ ID NO:1或2的LukA非變體序列，SEQ ID NO:3或4的LukA變體序列，SEQ ID NO:5或6的LukA變體序列，SEQ ID NO:7或8的LukA變體序列，SEQ ID NO:9或10的LukA變體序列，SEQ ID NO:11或12的LukA變體序列，或SEQ ID NO:13或14的LukA變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:19的LukB變體序列和SEQ ID NO:1或2的LukA非變體序列，SEQ ID NO:3或4的LukA變體序列，SEQ ID NO:5或6的LukA變體序列，SEQ ID NO:7或8的LukA變體序列，SEQ ID NO:9或10的LukA變體序列，SEQ ID NO:11或12的LukA變體序列，或SEQ ID NO:13或14的LukA變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:20的LukB變體序列和SEQ ID NO:1或2的LukA非變體序列，SEQ ID NO:3或4的LukA變體序列，SEQ ID NO:5或6的LukA變體序列，SEQ ID NO:7或8的LukA變體序列，SEQ ID NO:9或10的LukA變體序列，SEQ ID NO:11或12的LukA變體序列，或SEQ ID NO:13或14的LukA變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:21的LukB變體序列和SEQ ID NO:1或2的LukA非變體序列，SEQ ID NO:3或4的LukA變體序列，SEQ ID NO:5或6的LukA變體序列，SEQ ID NO:7或8的LukA變體序列，SEQ ID NO:9或10的LukA變體序列，SEQ ID NO:11或12的LukA變體序列，或SEQ ID NO:13或14的LukA變體序列。在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:22的LukB變體序列和SEQ ID NO:1或2的LukA非變體序列，SEQ ID NO:3或4的LukA變體序列，SEQ ID NO:5或6的LukA變體序列，SEQ ID NO:7或8的LukA變體序列，SEQ ID NO:9或10的LukA變體序列，SEQ ID NO:11或12的LukA變體序列，或SEQ ID NO:13或14的LukA變體序列。In any embodiment, the exemplary nucleic acid molecule of the present invention encodes the LukB variant sequence of SEQ ID NO:17 and the LukA non-variant sequence of SEQ ID NO:1 or 2, the LukA variant of SEQ ID NO:3 or 4 sequence, the LukA variant sequence of SEQ ID NO: 5 or 6, the LukA variant sequence of SEQ ID NO: 7 or 8, the LukA variant sequence of SEQ ID NO: 9 or 10, the LukA variant sequence of SEQ ID NO: 11 or 12 A LukA variant sequence, or a LukA variant sequence of SEQ ID NO: 13 or 14. In any embodiment, the exemplary nucleic acid molecules of the present invention encode the LukB variant sequence of SEQ ID NO: 18 and the LukA non-variant sequence of SEQ ID NO: 1 or 2, the LukA variant of SEQ ID NO: 3 or 4 sequence, the LukA variant sequence of SEQ ID NO: 5 or 6, the LukA variant sequence of SEQ ID NO: 7 or 8, the LukA variant sequence of SEQ ID NO: 9 or 10, the LukA variant sequence of SEQ ID NO: 11 or 12 A LukA variant sequence, or a LukA variant sequence of SEQ ID NO: 13 or 14. In any embodiment, the exemplary nucleic acid molecules of the present invention encode the LukB variant sequence of SEQ ID NO: 19 and the LukA non-variant sequence of SEQ ID NO: 1 or 2, the LukA variant of SEQ ID NO: 3 or 4 sequence, the LukA variant sequence of SEQ ID NO: 5 or 6, the LukA variant sequence of SEQ ID NO: 7 or 8, the LukA variant sequence of SEQ ID NO: 9 or 10, the LukA variant sequence of SEQ ID NO: 11 or 12 A LukA variant sequence, or a LukA variant sequence of SEQ ID NO: 13 or 14. In any embodiment, the exemplary nucleic acid molecule of the present invention encodes the LukB variant sequence of SEQ ID NO:20 and the LukA non-variant sequence of SEQ ID NO:1 or 2, the LukA variant of SEQ ID NO:3 or 4 sequence, the LukA variant sequence of SEQ ID NO: 5 or 6, the LukA variant sequence of SEQ ID NO: 7 or 8, the LukA variant sequence of SEQ ID NO: 9 or 10, the LukA variant sequence of SEQ ID NO: 11 or 12 A LukA variant sequence, or a LukA variant sequence of SEQ ID NO: 13 or 14. In any embodiment, the exemplary nucleic acid molecule of the present invention encodes the LukB variant sequence of SEQ ID NO:21 and the LukA non-variant sequence of SEQ ID NO:1 or 2, the LukA variant of SEQ ID NO:3 or 4 sequence, the LukA variant sequence of SEQ ID NO: 5 or 6, the LukA variant sequence of SEQ ID NO: 7 or 8, the LukA variant sequence of SEQ ID NO: 9 or 10, the LukA variant sequence of SEQ ID NO: 11 or 12 A LukA variant sequence, or a LukA variant sequence of SEQ ID NO: 13 or 14. In any embodiment, the exemplary nucleic acid molecule of the present invention encodes the LukB variant sequence of SEQ ID NO:22 and the LukA non-variant sequence of SEQ ID NO:1 or 2, the LukA variant of SEQ ID NO:3 or 4 sequence, the LukA variant sequence of SEQ ID NO: 5 or 6, the LukA variant sequence of SEQ ID NO: 7 or 8, the LukA variant sequence of SEQ ID NO: 9 or 10, the LukA variant sequence of SEQ ID NO: 11 or 12 A LukA variant sequence, or a LukA variant sequence of SEQ ID NO: 13 or 14.

在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:4的CC45 LukA變體序列和SEQ ID NO:16的CC45 LukB序列。編碼這種LukAB異二聚體(RARPR-15)的示例性核酸分子包含與SEQ ID NO:55 (CC45 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，該核苷酸序列可操作地與SEQ ID NO:59 (CC45 LukB)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列偶聯。編碼這種LukAB異二聚體的示例性核酸分子包含SEQ ID NO:55的核苷酸序列，其可操作地與SEQ ID NO:59的核苷酸序列偶聯。In any embodiment, an exemplary nucleic acid molecule of the invention encodes the CC45 LukA variant sequence of SEQ ID NO:4 and the CC45 LukB sequence of SEQ ID NO:16. Exemplary nucleic acid molecules encoding such LukAB heterodimers (RARPR-15) comprise at least 85%, at least 90%, at least 95%, at least A nucleotide sequence of 97% or at least 99% sequence similarity, which operably has at least 85%, at least 90%, at least 95% with the nucleotide sequence of SEQ ID NO:59 (CC45 LukB) , a nucleotide sequence coupling of at least 97% or at least 99% sequence similarity. An exemplary nucleic acid molecule encoding such a LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO:55 operably coupled to the nucleotide sequence of SEQ ID NO:59.

在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:4的CC45 LukA變體序列和SEQ ID NO:18的CC45 LukB變體序列。編碼這種LukAB異二聚體(RARPR-30)的示例性核酸分子包含與SEQ ID NO:55 (CC45 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與SEQ ID NO:61(CC45 LukB變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。編碼這種LukAB異二聚體的示例性核酸分子包含SEQ ID NO:55的核苷酸序列，其可操作地與SEQ ID NO:61的核苷酸序列偶聯。In any embodiment, an exemplary nucleic acid molecule of the invention encodes the CC45 LukA variant sequence of SEQ ID NO:4 and the CC45 LukB variant sequence of SEQ ID NO:18. An exemplary nucleic acid molecule encoding such a LukAB heterodimer (RARPR-30) comprises at least 85%, at least 90%, at least 95%, at least A nucleotide sequence of 97% or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 61 (CC45 LukB variant) having at least 85%, at least 90%, at least 95% , nucleotide sequences of at least 97% or at least 99% sequence similarity are operably coupled. An exemplary nucleic acid molecule encoding such a LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO:55 operably coupled to the nucleotide sequence of SEQ ID NO:61.

在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:3的CC8 LukA變體序列和SEQ ID NO:15的CC8 LukB序列。編碼這種LukAB異二聚體(RARPR-32)的示例性核酸分子包含與SEQ ID NO:54 (CC8 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與SEQ ID NO:58 (CC8 LukB)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。編碼這種LukAB異二聚體的示例性核酸分子包含SEQ ID NO:54的核苷酸序列，其可操作地與SEQ ID NO:58的核苷酸序列偶聯。In any embodiment, an exemplary nucleic acid molecule of the invention encodes the CC8 LukA variant sequence of SEQ ID NO:3 and the CC8 LukB sequence of SEQ ID NO:15. An exemplary nucleic acid molecule encoding such a LukAB heterodimer (RARPR-32) comprises at least 85%, at least 90%, at least 95%, at least A nucleotide sequence of 97% or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 58 (CC8 LukB) having at least 85%, at least 90%, at least 95%, at least Nucleotide sequences with 97% or at least 99% sequence similarity are operably coupled. An exemplary nucleic acid molecule encoding such a LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO:54 operably coupled to the nucleotide sequence of SEQ ID NO:58.

在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:3的CC8 LukA變體序列和SEQ ID NO:18的CC45 LukB變體序列。編碼這種LukAB異二聚體(RARPR-33)的示例性核酸分子包含與SEQ ID NO:54 (CC8 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與SEQ ID NO:61(CC45 LukB變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。編碼這種LukAB異二聚體的示例性核酸分子包含SEQ ID NO:54的核苷酸序列，其可操作地與SEQ ID NO:61的核苷酸序列偶聯。In any embodiment, an exemplary nucleic acid molecule of the invention encodes the CC8 LukA variant sequence of SEQ ID NO:3 and the CC45 LukB variant sequence of SEQ ID NO:18. An exemplary nucleic acid molecule encoding such a LukAB heterodimer (RARPR-33) comprises at least 85%, at least 90%, at least 95%, at least A nucleotide sequence of 97% or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 61 (CC45 LukB variant) having at least 85%, at least 90%, at least 95% , nucleotide sequences of at least 97% or at least 99% sequence similarity are operably coupled. An exemplary nucleic acid molecule encoding such a LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO:54 operably coupled to the nucleotide sequence of SEQ ID NO:61.

在任意的實施方案中，本發明的示例性核酸分子編碼SEQ ID NO:3的CC8 LukA變體序列和SEQ ID NO:17的CC8 LukB變體序列。編碼這種LukAB異二聚體(RARPR-34)的示例性核酸分子包含與SEQ ID NO:54 (CC8 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與SEQ ID NO:60(CC8 LukB變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。編碼這種LukAB異二聚體的示例性核酸分子包含SEQ ID NO:54的核苷酸序列，其可操作地與SEQ ID NO:60的核苷酸序列偶聯。In any embodiment, an exemplary nucleic acid molecule of the invention encodes the CC8 LukA variant sequence of SEQ ID NO:3 and the CC8 LukB variant sequence of SEQ ID NO:17. An exemplary nucleic acid molecule encoding such a LukAB heterodimer (RARPR-34) comprises at least 85%, at least 90%, at least 95%, at least A 97% or at least 99% sequence similarity to a nucleotide sequence of at least 85%, at least 90%, at least 95% to the nucleotide sequence of SEQ ID NO: 60 (CC8 LukB variant) , nucleotide sequences of at least 97% or at least 99% sequence similarity are operably coupled. An exemplary nucleic acid molecule encoding such a LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO:54 operably coupled to the nucleotide sequence of SEQ ID NO:60.

下表3提供了本發明的示例性核酸分子序列。Table 3 below provides exemplary nucleic acid molecule sequences of the invention.

表surface 3.3. 示例性Exemplary LukALuka 和and LukBLuk B 多核苷酸序列polynucleotide sequence 構建體名稱 construct name SEQ ID NO: SEQ ID NO: DNA序列 dna sequence CC8 LukAwt CC8 LukAwt 52 52 GCT CAC AAA GAT TCT CAG GAT CAA AAT AAG AAG GAG CAC GTC GAC AAG TCT CAG CAG AAA GAC AAG CGT AAT GTT ACA AAC AAG GAC AAA AAC AGC ACT GCT CCA GAC GAC ATT GGA AAA AAC GGT AAG ATT ACT AAA CGC ACC GAA ACG GTA TAT GAC GAA AAA ACG AAC ATT TTG CAA AAC TTG CAG TTC GAT TTC ATT GAC GAC CCC ACT TAT GAC AAG AAT GTC CTT CTG GTG AAG AAG CAG GGC AGC ATT CAC TCA AAC TTG AAA TTT GAG TCT CAC AAG GAG GAG AAG AAC TCC AAT TGG CTG AAA TAC CCA TCA GAG TAC CAC GTT GAT TTT CAA GTG AAA CGT AAC CGC AAA ACG GAA ATT TTG GAC CAA TTG CCG AAA AAC AAG ATC TCC ACC GCG AAA GTA GAC TCA ACA TTC AGT TAC TCT TCC GGC GGA AAG TTC GAC AGC ACT AAG GGG ATC GGG CGC ACT TCT TCC AAT TCG TAC TCG AAA ACG ATT TCT TAC AAT CAG CAG AAT TAT GAC ACT ATC GCA TCT GGT AAA AAT AAT AAC TGG CAC GTG CAT TGG TCG GTG ATT GCT AAT GAT TTA AAG TAT GGA GGT GAG GTA AAA AAT CGT AAT GAC GAG CTG CTG TTT TAC CGT AAC ACT CGC ATC GCA ACC GTT GAA AAC CCG GAA TTG TCC TTT GCC TCG AAA TAC CGC TAC CCT GCA TTA GTT CGT TCA GGC TTT AAT CCC GAG TTT TTG ACT TAT CTT TCC AAT GAA AAA TCG AAC GAG AAG ACT CAG TTC GAG GTT ACG TAC ACC CGC AAT CAG GAC ATT TTG AAG AAC CGT CCG GGA ATT CAC TAT GCG CCT CCC ATC TTA GAG AAG AAT AAG GAT GGA CAA CGT TTG ATC GTT ACA TAT GAA GTT GAC TGG AAA AAT AAG ACC GTA AAG GTT GTG GAT AAG TAT TCG GAT GAT AAT AAG CCC TAT AAA GAA GGG GCT CAC AAA GAT TCT CAG GAT CAA AAT AAG AAG GAG CAC GTC GAC AAG TCT CAG CAG AAA GAC AAG CGT AAT GTT ACA AAC AAG GAC AAA AAC AGC ACT GCT CCA GAC GAC ATT GGA AAA AAC GGT AAG ATT ACT AAA CGC ACC GAA ACG GTA TAT GAC GAA AAA ACG AAC ATT TTG CAA AAC TTG CAG TTC GAT TTC ATT GAC GAC CCC ACT TAT GAC AAG AAT GTC CTT CTG GTG AAG AAG CAG GGC AGC ATT CAC TCA AAC TTG AAA TTT GAG TCT CAC AAG GAG GAG AAG AAC TCC AAT TGG CTG AAA TAC CCA TCA GAG TAC CAC GTT GAT TTT CAA GTG AAA CGT AAC CGC AAA ACG GAA ATT TTG GAC CAA TTG CCG AAA AAC AAG ATC TCC ACC GCG AAA GTA GAC TCA ACA TTC AGT TAC TCT TCC GGC GGA AAG TTC GAC AGC ACT AAG GGG ATC GGG CGC ACT TCT TCC AAT TCG TAC TCG AAA ACG ATT TCT TAC AAT CAG CAG AAT TAT GAC ACT ATC GCA TCT GGT AAA AAT AAT AAC TGG CAC GTG CAT TGG TCG GTG ATT GCT AAT GAT TTA AAG TAT GGA GGT GAG GTA AAA AAT CGT AAT GAC GAG CTG CTG TTT TAC CGT AAC ACT CGC ATC GCA ACC GTT GAA AAC CCG GAA TTG TCC TTT GCC TCG AAA TAC CGC TAC CCT GCA TTA GTT CGT TCA GGC TTT AAT CCC GAG TTT TTG ACT TAT CTT TCC AAT GAA AAA TCG AAC GAG AAG ACT CAG TTC GAG GTT ACG TAC ACC CGC AAT CAG GAC ATT TTG AAG AAC CGT CCG GGA ATT CAC TAT GCG CCT CCC ATC TTA GAG AAG AAT AAG GAT GGA CAA CGT TTG ATC GTT ACA TAT GAA GTT GAC TGG AAA AAT AAG ACC GTA AAG GTT GTG GAT AAG TAT TCG GAT GAT AAT AAG CCC TAT AAA GAA GGG CC45 LukAwt CC45 LukAwt 53 53 GCG AAC AAA GAT TCT CAG GAC CAG ACC AAA AAG GAG CAC GTA GAC AAG GCC CAG CAA AAA GAG AAG CGT AAT GTG AAC GAC AAA GAT AAG AAT ACT CCG GGG CCA GAT GAT ATC GGC AAG AAC GGT AAA GTC ACG AAG CGT ACA GTG TCT GAG TAT GAC AAA GAA ACA AAC ATC CTG CAG AAC TTA CAA TTC GAC TTT ATT GAT GAT CCA ACT TAC GAT AAG AAT GTG TTG CTG GTT AAG AAA CAA GGT TCA ATC CAT TCT AAC TTG AAG TTC GAG TCA CAC CGT AAC GAA ACG AAC GCG TCG TGG TTG AAA TAT CCG TCA GAG TAT CAT GTT GAT TTT CAA GTA CAA CGT AAT CCC AAA ACG GAA ATT TTG GAC CAA TTA CCT AAA AAT AAG ATT AGC ACC GCC AAG GTT GAC TCA ACT TTC TCC TAC TCA TTA GGA GGA AAG TTC GAT TCG ACA AAA GGG ATC GGG CGT ACA TCT TCG AAT AGC TAC AGT AAG AGC ATT AGC TAT AAC CAG CAG AAC TAT GAT ACG ATT GCT TCA GGG AAA AAT AAC AAC CGT CAC GTA CAT TGG TCA GTG GTT GCG AAC GAT CTT AAA TAT GGA AAC GAG ATT AAG AAT CGT AAC GAC GAA TTT TTG TTT TAC CGC AAT ACA CGC CTT AGT ACC GTG GAA AAT CCC GAG CTG TCC TTC GCG TCG AAG TAT CGC TAT CCG GCC CTT GTG CGT TCG GGT TTC AAT CCC GAG TTC TTA ACA TAT ATT TCC AAT GAG AAA ACT AAC GAC AAG ACT CGC TTC GAA GTC ACC TAC ACT CGC AAC CAG GAC ATT CTG AAA AAC AAG CCT GGA ATT CAT TAC GGG CAA CCA ATT TTA GAG CAG AAT AAG GAT GGA CAG CGC TTT ATT GTG GTA TAT GAG GTG GAC TGG AAG AAT AAG ACA GTA AAA GTT GTG GAA AAG TAC TCT GAC CAG AAT AAG CCC TAT AAA GAA GGA GCG AAC AAA GAT TCT CAG GAC CAG ACC AAA AAG GAG CAC GTA GAC AAG GCC CAG CAA AAA GAG AAG CGT AAT GTG AAC GAC AAA GAT AAG AAT ACT CCG GGG CCA GAT GAT ATC GGC AAG AAC GGT AAA GTC ACG AAG CGT ACA GTG TCT GAG TAT GAC AAA GAA ACA AAC ATC CTG CAG AAC TTA CAA TTC GAC TTT ATT GAT GAT CCA ACT TAC GAT AAG AAT GTG TTG CTG GTT AAG AAA CAA GGT TCA ATC CAT TCT AAC TTG AAG TTC GAG TCA CAC CGT AAC GAA ACG AAC GCG TCG TGG TTG AAA TAT CCG TCA GAG TAT CAT GTT GAT TTT CAA GTA CAA CGT AAT CCC AAA ACG GAA ATT TTG GAC CAA TTA CCT AAA AAT AAG ATT AGC ACC GCC AAG GTT GAC TCA ACT TTC TCC TAC TCA TTA GGA GGA AAG TTC GAT TCG ACA AAA GGG ATC GGG CGT ACA TCT TCG AAT AGC TAC AGT AAG AGC ATT AGC TAT AAC CAG CAG AAC TAT GAT ACG ATT GCT TCA GGG AAA AAT AAC AAC CGT CAC GTA CAT TGG TCA GTG GTT GCG AAC GAT CTT AAA TAT GGA AAC GAG ATT AAG AAT CGT AAC GAC GAA TTT TTG TTT TAC CGC AAT ACA CGC CTT AGT ACC GTG GAA AAT CCC GAG CTG TCC TTC GCG TCG AAG TAT CGC TAT CCG GCC CTT GTG CGT TCG GGT TTC AAT CCC GAG TTC TTA ACA TAT ATT TCC AAT GAG AAA ACT AAC GAC AAG ACT CGC TTC GAA GTC ACC TAC ACT CGC AAC CAG GAC ATT CTG AAA AAC AAG CCT GGA ATT CAT TAC GGG CAA CCA ATT TTA GAG CAG AAT AAG GAT GGA CAG CGC TTT ATT GTG GTA TAT GAG GTG GAC TGG AAG AAT AAG ACA GTA AAA GTT GTG GAA AAG TAC TCT GAC CAG AAT AAG CCC TAT AAA GAA GGA CC8 LukAΔ10C CC8 LukAΔ10C 62 62 GCT CAC AAA GAT TCT CAG GAT CAA AAT AAG AAG GAG CAC GTC GAC AAG TCT CAG CAG AAA GAC AAG CGT AAT GTT ACA AAC AAG GAC AAA AAC AGC ACT GCT CCA GAC GAC ATT GGA AAA AAC GGT AAG ATT ACT AAA CGC ACC GAA ACG GTA TAT GAC GAA AAA ACG AAC ATT TTG CAA AAC TTG CAG TTC GAT TTC ATT GAC GAC CCC ACT TAT GAC AAG AAT GTC CTT CTG GTG AAG AAG CAG GGC AGC ATT CAC TCA AAC TTG AAA TTT GAG TCT CAC AAG GAG GAG AAG AAC TCC AAT TGG CTG AAA TAC CCA TCA GAG TAC CAC GTT GAT TTT CAA GTG AAA CGT AAC CGC AAA ACG GAA ATT TTG GAC CAA TTG CCG AAA AAC AAG ATC TCC ACC GCG AAA GTA GAC TCA ACA TTC AGT TAC TCT TCC GGC GGA AAG TTC GAC AGC ACT AAG GGG ATC GGG CGC ACT TCT TCC AAT TCG TAC TCG AAA ACG ATT TCT TAC AAT CAG CAG AAT TAT GAC ACT ATC GCA TCT GGT AAA AAT AAT AAC TGG CAC GTG CAT TGG TCG GTG ATT GCT AAT GAT TTA AAG TAT GGA GGT GAG GTA AAA AAT CGT AAT GAC GAG CTG CTG TTT TAC CGT AAC ACT CGC ATC GCA ACC GTT GAA AAC CCG GAA TTG TCC TTT GCC TCG AAA TAC CGC TAC CCT GCA TTA GTT CGT TCA GGC TTT AAT CCC GAG TTT TTG ACT TAT CTT TCC AAT GAA AAA TCG AAC GAG AAG ACT CAG TTC GAG GTT ACG TAC ACC CGC AAT CAG GAC ATT TTG AAG AAC CGT CCG GGA ATT CAC TAT GCG CCT CCC ATC TTA GAG AAG AAT AAG GAT GGA CAA CGT TTG ATC GTT ACA TAT GAA GTT GAC TGG AAA AAT AAG ACC GTA AAG GTT GTG GAT AAG TAT GCT CAC AAA GAT TCT CAG GAT CAA AAT AAG AAG GAG CAC GTC GAC AAG TCT CAG CAG AAA GAC AAG CGT AAT GTT ACA AAC AAG GAC AAA AAC AGC ACT GCT CCA GAC GAC ATT GGA AAA AAC GGT AAG ATT ACT AAA CGC ACC GAA ACG GTA TAT GAC GAA AAA ACG AAC ATT TTG CAA AAC TTG CAG TTC GAT TTC ATT GAC GAC CCC ACT TAT GAC AAG AAT GTC CTT CTG GTG AAG AAG CAG GGC AGC ATT CAC TCA AAC TTG AAA TTT GAG TCT CAC AAG GAG GAG AAG AAC TCC AAT TGG CTG AAA TAC CCA TCA GAG TAC CAC GTT GAT TTT CAA GTG AAA CGT AAC CGC AAA ACG GAA ATT TTG GAC CAA TTG CCG AAA AAC AAG ATC TCC ACC GCG AAA GTA GAC TCA ACA TTC AGT TAC TCT TCC GGC GGA AAG TTC GAC AGC ACT AAG GGG ATC GGG CGC ACT TCT TCC AAT TCG TAC TCG AAA ACG ATT TCT TAC AAT CAG CAG AAT TAT GAC ACT ATC GCA TCT GGT AAA AAT AAT AAC TGG CAC GTG CAT TGG TCG GTG ATT GCT AAT GAT TTA AAG TAT GGA GGT GAG GTA AAA AAT CGT AAT GAC GAG CTG CTG TTT TAC CGT AAC ACT CGC ATC GCA ACC GTT GAA AAC CCG GAA TTG TCC TTT GCC TCG AAA TAC CGC TAC CCT GCA TTA GTT CGT TCA GGC TTT AAT CCC GAG TTT TTG ACT TAT CTT TCC AAT GAA AAA TCG AAC GAG AAG ACT CAG TTC GAG GTT ACG TAC ACC CGC AAT CAG GAC ATT TTG AAG AAC CGT CCG GGA ATT CAC TAT GCG CCT CCC ATC TTA GAG AAG AAT AAG GAT GGA CAA CGT TTG ATC GTT ACA TAT GAA GTT GAC TGG AAA AAT AAG ACC GTA AAG GTT GTG GAT AAG TAT CC45 LukAΔ10C CC45 LukAΔ10C 63 63 GCAAATAAAGACTCTCAAGATCAGACTAAAAAGGAACATGTTGATAAGGCGCAACAAAAAGAAAAGCGTAATGTCAATGATAAGGACAAGAATACTCCGGGACCCGACGACATTGGCAAGAACGGAAAGGTGACAAAGCGTACCGTTAGTGAGTACGACAAGGAAACAAATATCCTGCAGAACTTACAGTTCGATTTTATTGACGATCCTACCTATGACAAGAATGTCCTGTTGGTGAAGAAACAGGGCAGCATTCATTCCAATTTAAAATTTGAAAGCCATCGTAACGAAACAAATGCATCTTGGCTTAAATACCCTTCTGAGTACCACGTAGATTTTCAGGTACAACGCAACCCAAAAACCGAAATTCTGGATCAACTGCCCAAGAATAAAATTTCTACGGCTAAAGTTGACAGTACATTTAGCTACAGTTTAGGGGGAAAGTTTGATAGTACAAAAGGAATTGGTCGTACTTCCAGTAACTCCTATTCGAAATCTATTTCCTATAATCAACAGAATTACGACACCATCGCATCCGGTAAAAACAATAATCGCCACGTACATTGGAGTGTTGTCGCGAATGACTTAAAGTACGGTAACGAAATCAAGAACCGCAACGACGAATTCTTATTCTATCGTAACACGCGTTTAAGCACCGTCGAGAACCCCGAGTTATCCTTTGCTAGCAAATATCGCTATCCTGCGTTAGTACGCTCAGGGTTCAATCCTGAGTTCTTAACCTACATCTCCAACGAGAAAACTAATGATAAGACACGCTTCGAGGTGACCTACACCCGTAATCAGGATATCCTTAAAAATAAACCGGGTATTCATTACGGGCAACCCATTTTAGAACAGAATAAGGACGGCCAACGTTTTATCGTGGTCTATGAGGTTGATTGGAAGAACAAGACAGTGAAAGTGGTTGAAAAGTAT GCAAATAAAGACTCTCAAGATCAGACTAAAAAGGAACATGTTGATAAGGCGCAACAAAAAGAAAAGCGTAATGTCAATGATAAGGACAAGAATACTCCGGGACCCGACGACATTGGCAAGAACGGAAAGGTGACAAAGCGTACCGTTAGTGAGTACGACAAGGAAACAAATATCCTGCAGAACTTACAGTTCGATTTTATTGACGATCCTACCTATGACAAGAATGTC CTGTTGGTGAAGAAACAGGGCAGCATTCATCCAATTTAAAATTTGAAAGCCATCGTAACGAAACAAATGCATCTTGGCTTAAATACCCCTTCTGAGTACCACGTAGATTTTCAGGTACAACGCAACCCAAAAACCGAAATTCTGGATCAACTGCCCAAGAATAAAATTTCTACGGCTAAAGTTGACAGTACATTTAGCTACAGTTTAGGGGGAAAGTTTGATAGTACAG AAAGGAATTGGTCGTACTTCCAGTAACTCCTATTCGAAATCTATTTCCTATAATCAACAGAATTACGACACCATCGCATCCGGTAAAAACAATAATCGCCACGTACATTGGAGTGTTGTCGCGAATGACTTAAAGTACGGTAACGAAATCAAGAACCGCAACGACGAATTCTTATTCTATCGTAACACGCGTTTAAGCACCGTCGAGAACCCCCGAGTTATTCCTTT GCTAGCAAATATCGCTATCCTGCGTTAGTACGCTCAGGGTTAATCCTGAGTTTCTTAACCTACATCTCCAACGAGAAAACTAATGATAAGACACGCTTCGAGGTGACCTACACCCGTAATCAGGATATCCTTAAAAATAAACCGGGTATTCATTACGGGCAACCCATTTTAGAACAGAATAAGGACGGCCAACGTTTTTATCGTGGTCTATGAGGTTGATTGGAAGAAC AAGACAGTGAAAGTGGTTGAAAAGTAT CC8 LukA W95 CC8 LukA W95 54 54 the CATAAAGATTCGCAGGATCAAAATAAGAAGGAGCATGTTGACAAGAGCCAGCAGAAAGACAAGCGCAATGTTACAAACAAAGATAAGAACTCTACAGCGCCCGATGACATTGGTAAGAACGGCAAGATAACTAAGCGGACGGAAACCGTGTATGACGAGAAAACTAACATTCTGCAAAATTTGCAATTTGACTTTATCGACGATCCAACCTATGACAAGAATGTCTTGCTTGTCAAAATGCAAGGTTCGATTCATTCAAACCTTAAATTTGAATCCCACAAAGAGGAGAAAAACTCTAATTGGTTAAAGTATCCTTCAGAATATCACATAGATTTCCAGGTAAAGAGAAACCGTAAAACGGAGATACTGGATCAACTGCCTAAAAACAAGATCTCGACAGCTAAGGTGGACGCTACGTTCTCGTACTCGTCTGGTGGGAAGTTCGACTCGACCAAAGGCATTGGGCGTACATCATCAAATAGCTATTCAAAGACTATTAGCTATAATCAGCAGAACTATGATACGATAGCTTCGGGTAAGAATAACAACTGGCACGTTCATTGGTCGATCATTGCAAATGACTTGAAGTATGGCGGAGAGGTAAAGAATCGCAACGATGAGCTGTTATTCTATCGCAATACGAGAATTGCGACTGTAGAGAACCCGGAATTGTCTTTTGCCTCCAAATATCGGTACCCGGCATTGGTACGCTCTGGTTTCAATCCTGAGTTTTTAACTTACCTTTCCAACGAAAAGAGTAATGAGAAGACCCAATTTGAGGTTACCTACACCCGTAACCAGGATATTTTGAAGAATCGGCCGGGCATCCATTATGCCCCACCAATCCTGGAGAAAAATAAAGACGGTCAGCGGCTTATTGTGACTTACGAGGTCGATTGGAAAAATAAGACGGTCAAGGTAGTGGACAAATATTCTGATGACAATAAACCGTACAAAGCTGGC CATAAAGATTCGCAGGATCAAAATAAGAAGGAGCATGTTGACAAGAGCCAGCAGAAAGACAAGCGCAATGTTACAAACAAAGATAAGAACTTCTACAGCGCCCGATGACATTGGTAAGAACGGCAAGATAACTAAGCGGACGGAAACCGTGTATGACGAGAAAACTAACATTCTGCAAAATTTGCAATTTGACTTTATCGACGATCCAACCTATGACAAGAATGT CTTGCTTGTCAAAATGCAAGGTTCGATTCATTCAAACCTTAAATTTGAATCCCACAAAGAGGAGAAAAACTCTAATTGGTTAAAGTATCCTCAGAATATCACATAGATTTCCAGGTAAAGAGAAACCGTAAAACGGAGATACTGGATCAACTGCCTAAAAACAAGATCTCGACAGCTAAGGTGGACGCTACGTTCTCGTACTCGTCTGGTGGGAAGTTCGACTCGACC AAAGGCATTGGGCGTACATCATCATCAAATAGCTATTCAAAGACTATTAGCTATAATCAGCAGAACTATGATACGATAGCTTCGGGTAAGAATAACAACTGGCACGTTCATTGGTCGATCATTGCAAATGACTTGAAGTATGGCGGAGAGGTAAAGAATCGCAACGATGAGCTGTTATTCTATCGCAATACGAGAATTGCGACTGTAGAGAACCCGGAATT GTCTTTTGCCTCCAAATATCGGTACCCGGCATTGGTACGCTCTGGTTTCAATCCTGAGTTTTTAACTTACCTTTCCAACGAAAAGAGTAATGAGAAGACCCAATTTGAGGTTACCTACACCCGTAACCAGGATATTTTGAAGAATCGGCCGGGCATCCATTATGCCCCACCAATCCTGGAGAAAAAATAAAGACGGTCAGCGGCTTATTGTGACTTACGAGG TCGATTGGAAAAATAAGACGGTCAAGGTAGTGGACAAATATTCTGATGACAATAAACCGTACAAAGCTGGC CC45 LukA W95 CC45 LukA W95 55 55 the GCTAATAAGGACTCCCAGGACCAGACAAAGAAGGAACACGTCGACAAAGCCCAGCAAAAAGAAAAACGCAACGTAAACGATAAGGACAAGAACACCCCAGGACCCGATGATATTGGGAAGAACGGTAAAGTCACAAAACGCACAGTGAGCGAGTACGATAAAGAAACAAATATCCTGCAAAATCTGCAATTTGACTTCATCGATGACCCTACCTATGATAAGAATGTGTTGTTGGTTAAGATGCAGGGAAGTATTCATTCCAACTTGAAATTCGAGAGCCACCGTAACGAAACGAATGCGAGTTGGTTAAAGTACCCTTCAGAATACCACATTGATTTTCAGGTGCAGCGTAACCCGAAAACCGAAATCTTAGACCAGCTGCCTAAAAACAAGATTTCTACGGCCAAGGTGGACGCAACTTTCAGTTATAGTCTTGGAGGAAAGTTCGACAGTACCAAAGGTATCGGCCGCACATCCTCAAACAGCTATTCGAAATCCATTTCTTACAACCAGCAAAATTATGACACGATCGCCTCAGGTAAGAACAACAATCGTCATGTGCATTGGAGCATCGTGGCTAACGATTTGAAATATGGTAACGAAATCAAAAATCGCAATGACGAGTTCTTGTTTTACCGCAATACTCGCCTTTCTACGGTAGAGAATCCTGAGCTTAGCTTTGCGAGCAAGTATCGTTACCCTGCTCTTGTACGTTCGGGTTTCAACCCAGAGTTCCTTACTTATATCTCCAATGAGAAGACGAACGATAAAACCCGTTTTGAAGTTACATACACGCGTAATCAGGACATCTTAAAGAATAAACCGGGGATTCATTATGGGCAGCCGATCTTAGAGCAAAATAAGGATGGACAGCGTTTCATTGTAGTGTATGAGGTTGACTGGAAGAACAAGACGGTAAAAGTAGTTGAAAAGTATTCCGACCAAAACAAGCCTTATAAGGCGGGT GCTAATAAGGACTCCCAGGACCAGACAAAGAAGGAACACGTCGACAAAGCCCAGCAAAAAGAAAAACGCAACGTAAACGATAAGGACAAGAACACCCCAGGACCCGATGATATTGGGAAGAACGGTAAAAGTCACAAAACGCACAGTGAGCGAGTACGATAAAGAAACAAATATCCTGCAAAATCTGCAATTTGACTTCATCGATGACCCTACCTATGATAAGAAT GTGTTGTTGGTTAAGATGCAGGGAAGTATTCATTCCAACTTGAAATTCGAGAGCCACCGTAACGAAACGAATGCGAGTTGGTTAAAGTACCTTCAGAATACCCATTGATTTTCAGGTGCAGCGTAACCCGAAAACCGAAATCTTAGACCAGCTGCCTAAAAACAAGATTTCTACGGCCAAGGTGGACGCAACTTTCAGTTATAGTCTTGGAGGAAAGTTCGA CAGTACCAAAGGTATCGGCCGCACATCCTCAAACAGCTATTCGAAATCCATTTCTTACAACCAGCAAAATTATGACACGATCGCCTCAGGTAAGAACAACAATCGTCATGTGCATTGGAGCATCGTGGCTAACGATTTGAAATATGGTAACGAAATCAAAAATCGCAATGACGAGTTCTTGTTTTACCGCAATACTCGCCTTTCTACGGTAGAGAATCCTGAGC TTAGCTTTGCGAGCAAGTATCGTTACCCCTGCTCTTGTACGTTCGGGTTTCAACCCAGAGTTCCTTACTTATATCTCCAATGAGAAGACGAACGATAAAACCCGTTTTGAAGTTACATACACGCGTAATCAGGACATCTTAAGAATAAACCGGGGATTCATTATGGGCAGCCGATCTTAGAGCAAAATAAGGATGGACAGCGTTTCATTGTAGTGTATGAGGTTGACTG GAAGAACAAGACGGTAAAAGTAGTTGAAAAGTATTCCGACCAAAAACAAGCTTATAAGGCGGGT CC8 LukA W97 W92 CC8 LukA W97 W92 56 56 CACAAAGACAGCCAGGATCAAAACAAGAAAGAGCACGTGGACAAGAGCCAGCAAAAGGATAAACGTAACGTTACCAACAAGGACAAAAACAGCACCGCGCCGGACGATATCGGCAAGAACGGCAAAATTACCAAGCGTACCGAGACCGTGTACGATGAAAAAACCAACATCCTGCAGAACCTGCAATTCGACTTTATTGACGATCCGACCTGCGATAAAAACGTGCTGCTGGTTAAGATGCAGGGCAGCATCCACAGCAACCTGAAATTCGAAAGCCACAAAGAGGAAAAGAACAGCAACTGGCTGAAGTACCCGAGCGAGTATCACATTGACTTTCAGGTGAAACGTAACCGTAAGACCGAAATCCTGGATCAACTGCCGAAGAACAAAATTAGCACCGCGAAGGTTTGCGCGACCTTCAGCTACAGCAGCGGTTGCAAATTTGACAGCACCAAGTGCATCGGCCGTACCAGCAGCAACAGCTATAGCAAAACCATCAGCTACAACCAGCAAAACTATGATACCATTGCGAGCGGCAAGAACAACAACTGGCACGTGCACTGGAGCATCATTGCGAACGACCTGAAATACGGTGGCGAGGTTAAGAACCGTAACGATGAACTGCTGTTCTATCGTAACACCCGTATCGCGACCGTGGAGAACCCGGAACTGAGCTTTGCGAGCAAATACCGTTATCCGGCGCTGGTGCGTAGCGGTTTCAACCCGGAGTTTCTGGTTTACCTGAGCAACGAGAAAAGCAACGAAAAGACCCAGTTCGAAGTTACCTACACCCGTAACCAAGACATCCTGAAGAACCGTCCGGGTATCCACTATGCTCCGCCGATTCTGGAGAAGAACAAAGATGGCCAACGTCTGATTGTGACCTATGAAGTTGACTGGAAGAACAAAACCGTTAAAGTGGTTGATAAGTACAGCGACGATAACAAACCGTATAAGGCGGGT CACAAAGACAGCCAGGATCAAAACAAGAAAGAGCACGTGGACAAGAGCCAGCAAAAGGATAAACGTAACGTTACCAACAAGGACAAAAACAGCACCGCGCCGGACGATATCGGCAAGAACGGCAAAATTACCAAGCGTACCGAGACCGTGTACGATGAAAAAACCAACATCCTGCAGAACCTGCAATTCGACTTTATTGACGATCCGACCTGCGATAAAAAC GTGCTGCTGGTTAAGATGCAGGGCAGCATCCACAGCAACCTGAAATTCGAAAGCCACAAAGAGGAAAAGAACAGCAACTGGCTGAAGTACCCGAGCGAGTATCACATTGACTTTCAGGTGAAACGTAACCGTAAGACCGAAATCCTGGAATCAACTGCCGAAGAACAAAATTAGCACCGCGAAGGTTTGCGCGACCTTCAGCTACAGCAGCGGTTGCAAATTTGACAGC ACCAAGTGCATCGGCCGTACCAGCAGCAACAGCTATAGCAAAACCATCAGTACAACCAGCAAACTATGATACCATTGCGAGCGGCAAGAACAACAACTGGCACGTGCACTGGAGCATCATTGCGAACGACCTGAAATACGGTGGCGAGGTTAAGAACCGTAACGATGAACTGCTGTTCTATCGTAACACCCGTATCGCGACCGTGGAGAACCCGGAACT GAGCTTTGCGAGCAAATACCGTTATCCGGCGCTGGTGCGTAGCGGTTTCAACCCGGAGTTTCTGGTTTACCTGAGCAACGAGAAAAGCAACGAAAAGACCCAGTTCGAAGTTACCTACACCCGTAACCAAAGACATCCTGAAGAACCGTCCGGGTATCCACTATGCTCCGCCGATTCTGGAGAAGAACAAAGATGGCCAACGTCTGATTGTGACCTATGAAGTTGA CTGGAAGAACAAAACCGTTAAAGTGGTTGATAAGTACAGCGACGATAACAAACCGTATAAGGCGGGT CC45 LukA W97 W92 CC45 LukA W97 W92 57 57 GCAAACAAAGACTCACAAGATCAGACAAAGAAAGAGCATGTAGACAAAGCTCAACAGAAGGAAAAGCGCAATGTGAACGACAAGGATAAAAATACTCCTGGTCCAGATGACATTGGTAAGAATGGTAAAGTTACTAAGCGGACCGTCTCTGAATATGATAAGGAGACAAATATTCTCCAGAATTTGCAATTCGATTTCATTGATGATCCGACGTGCGATAAGAACGTATTGCTCGTTAAAATGCAGGGCTCCATCCATTCGAATCTCAAGTTCGAATCCCATCGCAACGAGACAAACGCTTCCTGGCTCAAATATCCTAGCGAGTATCATATCGACTTCCAAGTTCAACGGAACCCTAAAACTGAAATCCTTGATCAACTCCCTAAGAACAAAATCTCAACTGCCAAGGTCTGTGCCACATTTTCTTATTCTCTTGGCTGCAAATTCGATTCAACAAAGTGTATTGGTCGTACATCAAGTAATAGCTATAGTAAAAGCATCAGTTATAACCAGCAAAACTATGATACAATCGCGTCAGGCAAAAACAATAATCGTCATGTCCATTGGTCCATTGTCGCGAACGACCTTAAGTACGGTAACGAAATTAAGAATCGGAACGATGAGTTTTTGTTCTATCGCAACACCCGTCTGTCTACTGTCGAAAACCCGGAGTTGTCCTTCGCAAGTAAATATCGCTATCCTGCTTTGGTACGTTCTGGGTTTAACCCGGAATTTCTCGTCTACATCAGCAACGAGAAAACAAATGACAAAACGCGCTTTGAAGTCACGTACACACGTAATCAGGACATCTTAAAAAATAAACCAGGGATTCACTATGGTCAGCCAATCTTGGAGCAGAATAAAGACGGCCAGCGTTTCATTGTCGTTTATGAAGTGGACTGGAAAAACAAAACTGTTAAGGTGGTTGAGAAATATTCCGACCAAAACAAACCGTATAAGGCCGGT GCAAACAAAGACTCACAAGATCAGACAAAGAAAGAGCATGTAGACAAAAGCTCAACAGAAGGAAAAGCGCAATGTGAACGACAAGGATAAAAATACTCCTGGTCCAGATGACATTGGTAAGAATGGTAAAGTTACTAAGCGGACCGTCTCTGAATATGATAAGGAGACAAATATTTCCAGAATTTGCAATTCGATTTCATTGATGATCCGACGTGCGATAAGAACGT ATTGCTCGTTAAAATGCAGGGCTCCATCCATTCGAATCTCAAGTTCGAATCCCATCGCAACGAGACAAACGCTTCCTGGCTCAAATATCCTAGCGAGTATCATATCGACTTCCAAGTTCAACGGAACCCTAAAACTGAAATCCTTGATCAACTCCCTAAGAACAAAATCTCAACTGCCAAGGTCTGTGCCACATTTTCTTATTCTCTTGGCTGCAAATTCGATTCAACAAA GTGTATTGGTCGTACATCAAGTAATAGCTATAGTAAAAGCATCAGTTATAACCAGCAAAACTATGATACAATCGCGTCAGGCAAAAACAATAATCGTCATGTCCATTGGTCCATTGTCGCGAACGACCTTAAGTACGGTAACGAAATTAAGAATCGGAACGATGAGTTTTTTGTTTCTATCGCAACACCCGTCTGTCTACTGTCGAAAACCCGGAGTTGTCCTTC GCAAGTAAATATCGCTATCCTGCTTTGGTACGTTCTGGGTTTAACCCGGAATTTCTCGTCTACATCAGCAACGAGAAAACAAATGACAAAACGCGCTTTGAAGTCACGTACACACGTAATCAGGACATTCTTAAAAATAAACCAGGGATTCACTATGGTCAGCCAAATCTTGGAGCAGAATAAAAGACGGCCAGCGTTTCATTGTCGTTTATGAAGTGGACTGGAA AAACAAAACTGTTAAGGTGGTTGAGAAATATTCCGACCAAAACAAACCGTATAAGGCCGGT CC8 LukBwt CC8 LukBwt 58 58 the AAAATCAATTCTGAAATTAAGCAAGTGTCCGAAAAAAATTTGGATGGAGACACGAAGATGTATACGCGTACTGCTACGACGTCAGACTCCCAGAAGAACATTACACAGAGTCTGCAATTTAATTTTCTGACAGAACCAAACTATGACAAGGAAACTGTCTTTATTAAGGCTAAAGGGACTATCGGAAGCGGCTTACGCATTTTAGACCCCAACGGTTATTGGAATAGCACGCTGCGCTGGCCGGGCAGTTACTCAGTATCAATCCAAAATGTCGATGATAACAATAACACCAATGTTACCGATTTCGCCCCCAAGAACCAGGATGAATCGCGCGAGGTTAAATACACATACGGCTACAAGACAGGCGGTGACTTTAGCATCAACCGTGGGGGCTTGACAGGGAATATTACTAAGGAATCAAATTATAGTGAGACTATCTCTTATCAACAACCGTCCTATCGTACCTTATTAGACCAGAGTACCTCCCACAAAGGTGTAGGGTGGAAAGTTGAAGCGCACCTGATTAATAATATGGGTCACGATCACACACGCCAACTGACCAACGACAGTGACAACCGCACAAAAAGTGAAATTTTTAGTCTTACCCGTAACGGAAATCTGTGGGCCAAAGACAATTTTACACCGAAAGATAAGATGCCGGTCACTGTATCTGAGGGGTTCAATCCCGAGTTTTTAGCAGTAATGTCGCATGACAAAAAGGACAAAGGGAAATCCCAGTTTGTTGTCCACTATAAGCGTAGCATGGATGAATTCAAAATCGACTGGAACCGTCACGGTTTCTGGGGTTACTGGTCAGGTGAGAACCACGTAGACAAGAAAGAGGAGAAACTGAGCGCATTATATGAGGTTGATTGGAAAACGCACAATGTGAAATTTGTTAAAGTCCTGAATGACAACGAGAAAAAG AAAATCAATTCTGAAATTAAGCAAGTGTCCGAAAAAATTTGGATGGAGACACGAAGATGTATACGCGTACTGCTACGACGTCAGACTCCCAGAAGAACATTACACAGAGTCTGCAATTTAATTTTCTGACAGAACCAAACTATGACAAGGAAACTGTCTTTTATTAAGGCTAAAGGGACTATCGGAAGCGGCTTACGCATTTTAGACCCCAACGGTTATTGGA ATAGCACGCTGCGCTGGCCGGGCAGTTACTCAGTATCAATCCAAAATGTCGATGATAACAATAACACCAATGTTACCGATTTCGCCCCCAAGAACCAGGATGAATCGCGCGAGGTTAAATACACATACGGCTACAAGACAGGCGGTGACTTTAGCATCAACCGTGGGGGCTTGACAGGGAATATTACTAAGGAATCAAAATTATAGTGAGACTATCTCTTAT CAACAACCGTCCTATCGTACCTTATTAGACCAGAGTACCTCCCAAAGGTGTAGGGTGGAAAGTTGAAGCGCACCTGATTAATAATATGGGTCACGATCACACACGCCAACTGACCAACGACAGTGACAACCGCCACAAAAGTGAAATTTTTAGTCTTACCCGTAACGGAAATCTGTGGGCCAAAGACAATTTTACACCGAAAGATAAGATGCCGGTCACTGTAT CTGAGGGGTTTCAATCCCGAGTTTTTAGCAGTAATGTCGCATGACAAAAAAGGACAAAGGGAAATCCCAGTTTGTTGTCCACTATAAGCGTAGCATGGATGAATTCAAAATCGACTGGAACCGTCACGGTTTCTGGGGTTACTGGTCAGGTGAGAACCACGTAGACAAGAAAGAGGAGAAACTGAGCGCATTATATGAGGTTGATTGGAAAACGCACA ATGTGAAATTTGTTAAAGTCCTGAATGACAACGAGAAAAAAG CC45 LukBwt CC45 LukBwt 59 59 the GAAATTAAGTCTAAGATCACAACAGTATCGGAGAAAAACCTGGATGGCGATACTAAGATGTATACACGCACCGCCACTACTTCGGACACGGAGAAGAAGATCTCACAATCGTTACAGTTTAATTTTCTTACAGAACCGAACTACGACAAAGAGACCGTCTTCATTAAAGCTAAAGGTACGATTGGTTCGGGATTAAAAATTCTGAATCCGAATGGCTATTGGAACAGTACCTTACGTTGGCCGGGGTCATATTCTGTATCCATTCAAAACGTGGACGACAATAACAACAGCACCAATGTGACAGATTTCGCTCCAAAGAATCAGGATGAGTCCCGCGAGGTGAAATATACCTATGGGTACAAAACAGGAGGTGACTTTAGCATTAACCGTGGTGGCTTGACTGGTAATATCACGAAGGAAAAAAATTACTCTGAGACTATTTCCTACCAACAGCCGTCGTATCGCACCTTGATCGACCAACCAACGACTAACAAAGGGGTCGCGTGGAAAGTTGAGGCCCACAGTATTAACAATATGGGCCACGATCACACTCGTCAGCTTACTAACGATTCGGATGACCGCGTCAAGTCGGAAATTTTCAGCCTGACGCGTAACGGAAATTTGTGGGCTAAAGACAATTTCACTCCTAAGAACAAGATGCCCGTGACTGTTTCCGAAGGCTTTAATCCCGAATTCTTAGCGGTGATGTCTCATGATAAAAATGATAAAGGAAAATCGCGCTTCATTGTGCATTATAAGCGTTCTATGGACGACTTCAAATTGGATTGGAATAAGCACGGATTCTGGGGGTACTGGTCCGGGGAAAATCACGTAGATCAAAAGGAAGAGAAGTTGTCCGCTTTGTATGAAGTGGACTGGAAGACTCACGACGTTAAGTTGATCAAGACCTTCAATGACAAAGAGAAGAAA GAAATTAAGTCTAAGATCACAACAGTATCGGAGAAAAAACCTGGATGGCGATACTAAGATGTATACACGCACCGCCACTACTTCGGACACGGAGAAGAAGATCTCACAATCGTTACAGTTTAATTTTCTTACAGAACCGAACTACGACAAAGAGACCGTCTTCATTAAAGCTAAAGGTACGATTGGTTCGGGATTAAAATTCTGAATCCGAATGGCTATTGGA ACAGTACCTTACGTTGGCCGGGGTCATATTCTGTATCCATTCAATTCAAAACGTGGACGACAATAACACAGCACCAATGTGACAGATTTCGCTCCAAAGAATCAGGATGAGTCCCGCGAGGTGAAATATACCTATGGGTACAAAACAGGAGGTGACTTTTAGCATTAACCGTGGTGGCTTGACTGGTAATATCACGAAGGAAAAAAAATTACTCTGAGACTATTTCCT ACCAACAGCCGTCGTATCGCACCTTGATCGACCAACCAACGACTAACAAAGGGGTCGCGTGGAAAGTTGAGGCCCACAGTATTAACAATATGGGCCACGATCACACTCGTCAGCTTACTAACGATTCGGATGACCGCGTCAAGTCGGAAATTTTCAGCCTGACGCGTAACGGAAATTTGTGGGCTAAAGACAATTTCACTCCTAAGAACAAGATGCCCGTGA CTGTTTCCGAAGGCTTTAATCCCGAATTCTTAGCGGTGATGTCTCATGATAAAAATGATAAAGGAAAATCGCGCTTCATTGTGCATTATAAGCGTTCTATGGACGACTTCAAATTGGATTGGAATAAGCACGGATTCTGGGGGTACTGGTCCGGGGAAAATCACGTAGATCAAAGGAAGAGAAGTTGTCCGCTTTGTATGAAGTGGACTGAAG ACTCACGACGTTAAGTTGATCAAGACCTTCAATGACAAAGAGAAGAAA CC8 LukB Val53Leu CC8 LukB Val53Leu 60 60 the AAGATCAATTCGGAAATTAAACAGGTAAGTGAGAAAAATTTGGATGGCGATACCAAAATGTACACCCGCACCGCTACCACGTCAGATTCACAAAAAAATATTACACAGTCCTTGCAGTTCAATTTCCTGACAGAACCGAATTACGACAAGGAGACTTTGTTCATTAAAGCCAAGGGAACCATCGGGTCCGGATTGCGTATCTTGGACCCGAACGGATATTGGAACTCGACCTTACGTTGGCCGGGGTCTTACAGTGTTAGTATCCAAAACGTAGATGATAACAATAACACAAACGTGACAGATTTTGCACCTAAAAACCAGGACGAAAGCCGCGAGGTAAAGTACACATATGGGTATAAAACAGGGGGGGACTTTTCCATCAACCGTGGTGGTTTGACCGGGAACATCACCAAAGAGTCAAATTACAGTGAGACCATCAGTTATCAGCAGCCGTCCTATCGTACATTATTGGATCAGTCGACTTCACATAAAGGGGTCGGATGGAAAGTAGAGGCTCATTTGATCAACAACATGGGTCACGATCATACACGTCAGTTAACGAACGATAGCGATAATCGCACGAAGTCAGAAATCTTTAGTCTGACTCGTAACGGTAACTTGTGGGCCAAGGACAATTTCACGCCCAAAGATAAGATGCCTGTGACGGTATCGGAGGGGTTCAATCCAGAATTCCTTGCTGTAATGTCCCATGACAAAAAAGACAAGGGCAAATCGCAATTTGTAGTCCACTATAAGCGTTCTATGGACGAGTTCAAGATTGACTGGAACCGCCACGGCTTCTGGGGGTACTGGAGTGGTGAGAATCATGTGGATAAAAAGGAGGAGAAACTTAGCGCCCTGTATGAGGTAGATTGGAAAACACACAATGTCAAGTTCGTGAAAGTTCTTAATGACAACGAAAAAAAA AAGATCAATTCGGAAATTAAACAGGTAAGTGAGAAAAATTTGGATGGCGATACCAAAATGTACACCCGCACCGCTACCACGTCAGATTCACAAAAAAATATTACACAGTCCTTGCAGTTCAATTTCCTGACAGAACCGAATTACGACAAGGAGACTTTGTTCATTAAAGCCAAGGGAACCATCGGGTCCGGATTGCGTATCTTGGACCCGAACGGATATTGGA ACTCGACCTTACGTTGGCCGGGGTCTTACAGTGTTAGTATCCAAAACGTAGATGATAACAATAACACAAACGTGACAGATTTTGCACCTAAAAACCAGGACGAAAGCCGCGAGGTAAAGTACACATATGGGTATAAAACAGGGGGGGACTTTTCCATCAACCGTGGTGGTTTGACCGGGAACATCACCAAAGAGTCAAATTACAGTGAGACCATCAGTTC AGCAGCCGTCCTATCGTACATTATTGGATCAGTCGACTTCACATAAAGGGGTCGGATGGAAAGTAGAGGCTCATTTGATCAACAACATGGGTCACGATCATACACGTCAGTTAACGAACGATAGCGATAATCGCACGAAGTCAGAAATCTTTTAGTCTGACTCGTAACGGTAACTTGTGGGCCAAGGACAATTGTGGGCCAAGGACAATTTCACGCCCAAAGATAAGATGCCTGTGACGG TATCGGAGGGGTTCAATCCAGAATTCCTTGCTGTAATGTCCCATGACAAAAAAAGACAAGGGCAAATCGCAATTTGTAGTCCACTATAAGCGTTCTATGGACGAGTTCAAAGATTGACTGGAACCGCCACGGCTTCTGGGGGTACTGGAGTGGTGAGAATCATGTGGATAAAAAGGAGGAGAAACTTAGCGCCCTGTATGAGGTAGATTGGAAA ACACACAATGTCAAGTTCGTGAAAGTTCTTAATGACAACGAAAAAAAAA CC45 LukB Val53Leu CC45 LukB Val53Leu 61 61 the GAGATCAAGAGCAAAATTACCACCGTGAGCGAAAAGAACCTGGACGGTGATACCAAAATGTATACCCGTACCGCGACCACCAGCGACACCGAGAAGAAAATTAGCCAGAGCCTGCAATTCAACTTTCTGACCGAGCCGAACTACGATAAGGAAACCCTGTTCATCAAGGCGAAAGGCACCATTGGTAGCGGCCTGAAAATCCTGAACCCGAACGGTTATTGGAACAGCACCCTGCGTTGGCCGGGTAGCTACAGCGTGAGCATCCAGAACGTTGACGATAACAACAACAGCACCAACGTGACCGACTTCGCGCCGAAGAACCAAGATGAGAGCCGTGAAGTTAAATACACCTATGGTTACAAAACCGGTGGCGACTTTAGCATTAACCGTGGTGGCCTGACCGGCAACATCACCAAGGAGAAAAACTATAGCGAAACCATTAGCTATCAGCAACCGAGCTACCGTACCCTGATCGATCAGCCGACCACCAACAAGGGTGTGGCGTGGAAAGTTGAGGCGCACAGCATTAACAACATGGGCCACGACCACACCCGTCAACTGACCAACGATAGCGACGATCGTGTGAAGAGCGAAATCTTCAGCCTGACCCGTAACGGTAACCTGTGGGCGAAAGACAACTTTACCCCGAAGAACAAAATGCCGGTGACCGTTAGCGAGGGTTTCAACCCGGAATTTCTGGCGGTGATGAGCCACGACAAGAACGATAAGGGCAAAAGCCGTTTCATTGTTCACTACAAACGTAGCATGGACGATTTCAAGCTGGACTGGAACAAACACGGTTTTTGGGGCTATTGGAGCGGCGAGAACCACGTTGATCAGAAAGAGGAGAAACTGAGCGCGCTGTACGAAGTGGACTGGAAGACCCACGATGTTAAGCTGATCAAAACCTTTAACGATAAAGAAAAGAAA GAGATCAAGAGCAAAATTACCACCGTGAGCGAAAAGAACCTGGACGGTGATACCAAAATGTATACCCGTACCGCGACCACCAGCGACACCGAGAAGAAAATTAGCCAGAGCCTGCAATTCAACTTTCTGACCGAGCCGAACTACGATAAGGAAACCCTGTTCATCAAGGCGAAAGGCACCATTGGTAGCGGCCTGAAAATCCTGAACCCGAACGGTTATTGGAAC AGCACCCTGCGTTGGCCGGGTAGCTACAGCGTGAGCATCCAGAACGTTGACGATAACAACAACAGCACCAACGTGACCGACTTCGCGCCGAAGAACCAAGATGAGAGCCGTGAAGTTAAATACACCTATGGTTACAAACCGGTGGCGACTTTAGCATTAACCGTGGTGGCCTGACCGGCAACATCACCAAGGAGAAAAACTATAGCGAAACCATTAGCTAAG CAACCGAGCTACCGTACCCTGATCGATCAGCCGACCACCAACAAGGGTGTGGCGTGGAAAGTTGAGGCCACAGCATTAACAACATGGGCCACGACCACACCCGTCAACTGACCAACGATAGCGACGATCGTGTGAAGAGCGAAATCTTCAGCCTGACCCGTAACGGTAACCTGTGGGCGAAAGACAACTTTACCCCCGAAGAACAAAATGCCGGTGACCGTTA GCGAGGGTTTCAACCCGGAATTTCTGGCGGTGATGAGCCACGACAAGAACGATAAGGGCAAAAGCCGTTTCATTGTTCACTACAAACGTAGCATGGACGATTTCAAGCTGGACTGGAACAAACACGGTTTTTGGGGCTATTGGAGCGGCGAGAACCACGTTGATCAGAAAGAGGAGAAACTGAGCGCGCTGTACGAAGTGGACTGGAAGACCCACG ATGTTAAAGCTGATCAAAACCTTTAACGATAAAGAAAAAGAAA

在任意的實施方案中，編碼如本文所述的編碼變異LukA和LukB多肽的核酸分子被密碼子優化以在哺乳動物細胞，優選人細胞中進行表達。密碼子優化的方法是已知的，並且已經在前面描述過(例如授予Seed的國際專利申請公開號WO1996/09378，其全部內容通過引用結合於此)。如果與野生型序列相比，至少一個非優選密碼子被更優選的密碼子取代，則序列被認為是密碼子優化的。這裏，非優選密碼子是在生物體中使用頻率低於編碼相同氨基酸的另一個密碼子的密碼子，更優選的密碼子是在生物體中使用頻率高於非優選密碼子的密碼子。用於特定生物體的密碼子使用頻率可以在本領域公知的和可獲得的密碼子頻率表中找到。優選多於一個非優選密碼子，例如多於10%、40%、60%、80%的非優選密碼子，優選大多數(例如，至少90%)或所有非優選密碼子被更優選的密碼子替換。優選地，生物體中最常用的密碼子用於密碼子優化的序列。優選密碼子的替換通常導致更高的表達。In any embodiment, nucleic acid molecules encoding variant LukA and LukB polypeptides encoding variants as described herein are codon-optimized for expression in mammalian cells, preferably human cells. Methods of codon optimization are known and have been described previously (eg, International Patent Application Publication No. WO 1996/09378 to Seed, the entire contents of which are hereby incorporated by reference). A sequence is considered codon-optimized if at least one non-preferred codon is replaced by a more preferred codon compared to the wild-type sequence. Here, a non-preferred codon is a codon that is used less frequently in an organism than another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. Codon usage frequencies for a particular organism can be found in codon frequency tables well known and available in the art. More than one non-preferred codon is preferred, e.g., more than 10%, 40%, 60%, 80% of the non-preferred codons, preferably most (e.g., at least 90%) or all of the non-preferred codons are replaced by more preferred codons sub-replacement. Preferably, the most commonly used codons in the organism are used for the codon-optimized sequence. Substitution of preferred codons generally results in higher expression.

本發明的多核苷酸序列可以使用常規分子生物學技術來進行克隆，或者通過DNA合成從頭生成，這可以由在DNA合成和/或分子克隆領域有業務的服務公司(例如GeneArt、GenScript、Invitrogen、Eurofins)使用常規程序來進行。The polynucleotide sequences of the present invention can be cloned using conventional molecular biology techniques, or generated de novo by DNA synthesis, which can be provided by service companies operating in the field of DNA synthesis and/or molecular cloning (e.g., GeneArt, GenScript, Invitrogen, Eurofins) using conventional procedures.

在任意的實施方案中，將前述核酸分子***載體，即用於本文所述疫苗組合物的表達載體。或者，可以將這些核酸分子***到表達載體中，該表達載體被轉化或轉染到合適的宿主細胞中，用於表達和分離編碼的變異LukA蛋白、變異LukB蛋白或變異LukB複合物(作為穩定的異二聚體)，其中變異LukB複合物包含本文公開的變異LukA和非變異LukB、非變異LukA和變異LukB、或變異LukA和變異LukB。In any of the embodiments, the aforementioned nucleic acid molecule is inserted into a vector, ie, an expression vector for use in the vaccine compositions described herein. Alternatively, these nucleic acid molecules can be inserted into expression vectors, which are transformed or transfected into suitable host cells for expression and isolation of encoded variant LukA proteins, variant LukB proteins or variant LukB complexes (as stable heterodimer), wherein the variant LukB complex comprises variant LukA and non-variant LukB, non-variant LukA and variant LukB, or variant LukA and variant LukB disclosed herein.

根據本發明的這一方面，編碼本文所述的金黃色葡萄球菌LukA和/或LukB蛋白及其多肽的核酸分子可以加入到能夠表達由核酸序列構建體編碼的LukA和/或LukB蛋白或多肽的任何表達載體中。合適的表達載體包含控制、調節、引起或允許由這種載體編碼的LukA和/或LukB蛋白或多肽表達的核酸序列要素。這些要素可以包括轉錄增強子結合位點、RNA聚合酶起始位點、核糖體結合位點和其他促進編碼多肽在給定表達系統中表達的位點。合適的載體包括但不限於DNA載體、質粒載體、線性核酸和病毒載體，例如腺病毒載體。According to this aspect of the present invention, the nucleic acid molecule of coding Staphylococcus aureus LukA and/or LukB protein and polypeptide thereof described herein can be added to can express the LukA and/or LukB protein or polypeptide encoded by nucleic acid sequence construct in any expression vector. Suitable expression vectors comprise nucleic acid sequence elements that control, regulate, cause or permit expression of the LukA and/or LukB proteins or polypeptides encoded by such vectors. These elements may include transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites and other sites that facilitate expression of the encoded polypeptide in a given expression system. Suitable vectors include, but are not limited to, DNA vectors, plasmid vectors, linear nucleic acids, and viral vectors, such as adenoviral vectors.

在一個實施方案中，所述表達載體是環形質粒(參見，例如，Muthumani等人，“Optimized and Enhanced DNA Plasmid Vector Based In vivo Construction of a Neutralizing anti-HIV-1 Envelope Glycoprotein Fab,” Hum. Vaccin. Immunother. 9: 2253-2262 (2013)，其全部內容通過引用結合於此)。質粒可以通過整合到細胞基因組中來轉化靶細胞，或者存在於染色體外(例如，具有複制起點的自主複制質粒)。示例性的質粒載體包括但不限於pCEP4、pREP4、pVAX、pcDNA3.0、provax或能夠表達由重組核酸序列構建體編碼的變異LukA和/或變異LukB蛋白或多肽的任何其他質粒表達載體。In one embodiment, the expression vector is a circular plasmid (see, e.g., Muthumani et al., "Optimized and Enhanced DNA Plasmid Vector Based In vivo Construction of a Neutralizing anti-HIV-1 Envelope Glycoprotein Fab," Hum. Vaccin. Immunother. 9: 2253-2262 (2013), the entire contents of which are hereby incorporated by reference). Plasmids can transform target cells by integrating into the cellular genome, or exist extrachromosomally (eg, autonomously replicating plasmids with origins of replication). Exemplary plasmid vectors include, but are not limited to, pCEP4, pREP4, pVAX, pcDNA3.0, provax, or any other plasmid expression vector capable of expressing variant LukA and/or variant LukB proteins or polypeptides encoded by recombinant nucleic acid sequence constructs.

在另一個實施方案中，表達載體是線性表達盒(“LEC”)。LEC能夠通過電穿孔被有效地遞送至受試者，以表達由本文所述重組核酸分子編碼的LukA和/或LukB蛋白或多肽。LEC可以是任何沒有磷酸骨架的線性DNA。在一個實施方案中，LEC不包含任何抗生素抗性基因和/或磷酸骨架。在另一個實施方案中，LEC不包含與所需基因表達無關的其他核酸序列。In another embodiment, the expression vector is a linear expression cassette ("LEC"). LECs can be efficiently delivered to a subject by electroporation to express LukA and/or LukB proteins or polypeptides encoded by the recombinant nucleic acid molecules described herein. LEC can be any linear DNA without a phosphate backbone. In one embodiment, the LEC does not comprise any antibiotic resistance genes and/or phosphate backbone. In another embodiment, the LEC does not contain other nucleic acid sequences unrelated to desired gene expression.

LEC可以來自任何能夠線性化的質粒。該質粒能夠表達本文所述重組核酸分子編碼的LukA和/或LukB蛋白或多肽。示例性質粒包括但不限於pNP(Puerto Rico/34)、pM2(New Caledonia/99)、WLV009、pVAX、pcDNA3.0或provax，或能夠表達由重組核酸序列構建體編碼的變異LukA和/或變異LukB蛋白或多肽的任何其他表達載體。LECs can be derived from any plasmid capable of linearization. The plasmid is capable of expressing the LukA and/or LukB protein or polypeptide encoded by the recombinant nucleic acid molecule described herein. Exemplary plasmids include, but are not limited to, pNP (Puerto Rico/34), pM2 (New Caledonia/99), WLV009, pVAX, pcDNA3.0 or provax, or capable of expressing variant LukA and/or variants encoded by recombinant nucleic acid sequence constructs Any other expression vector of LukB protein or polypeptide.

在另一個實施方案中，所述表達載體是病毒載體。能夠表達LukA和/或LukB蛋白或多肽的合適的病毒載體包括，例如，腺相關病毒(AAV)載體(參見，例如，Krause等人，“Delivery of Antigens by Viral Vectors for Vaccination,” Ther. Deliv. 2(1):51-70 (2011)；Ura等人，“Developments in Viral Vector-Based Vaccines,” Vaccines 2: 624-641 (2014)； Buning等人，"Recent Developments in Adeno- associated Virus Vector Technology," J. Gene Med. 10:717-733 (2008)，其中每一篇都在此全文引入作為參考)，慢病毒載體(參見，例如，Ura等人，“Developments in Viral Vector-Based Vaccines,” Vaccines 2: 624-641 (2014)；和Hu等人，“Immunization Delivered by Lentiviral Vectors for Cancer and Infection Diseases,” Immunol. Rev. 239: 45-61 (2011)，其在此全文引入作為參考)、逆轉錄病毒載體(例如參見Ura等人，“Developments in Viral Vector-Based Vaccines,” Vaccines 2: 624-641 (2014)，其在此全文引入作為參考)、牛痘病毒、複制缺陷型腺病毒載體和無骨腺病毒載體（gutless adenovirus vector）(例如參見美國專利No. 5,872,005，其全部內容通過引用結合於此)。用於產生和分離適合作為載體的腺相關病毒 (AAVs) 的方法在本領域是已知的(參見，例如，Grieger & Samulski，"Adeno-associated Virus as a Gene Therapy Vector: Vector Development, Production and Clinical Applications," Adv. Biochem. Engin/Biotechnol. 99: 119-145 (2005)，Buning等人，"Recent Developments in Adeno- associated Virus Vector Technology," J. Gene Med. 10:717-733 (2008)，其中每一篇都通過引用整體結合於此)。In another embodiment, the expression vector is a viral vector. Suitable viral vectors capable of expressing LukA and/or LukB proteins or polypeptides include, for example, adeno-associated virus (AAV) vectors (see, e.g., Krause et al., "Delivery of Antigens by Viral Vectors for Vaccination," Ther. Deliv. 2(1):51-70 (2011); Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2: 624-641 (2014); Buning et al., "Recent Developments in Adeno-associated Virus Vector Technology ," J. Gene Med. 10:717-733 (2008), each of which is hereby incorporated by reference in its entirety), lentiviral vectors (see, e.g., Ura et al., "Developments in Viral Vector-Based Vaccines, Vaccines 2: 624-641 (2014); and Hu et al., "Immunization Delivered by Lentiviral Vectors for Cancer and Infection Diseases," Immunol. Rev. 239: 45-61 (2011), which are hereby incorporated by reference in their entirety) , retroviral vectors (see, e.g., Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2: 624-641 (2014), which is hereby incorporated by reference in its entirety), vaccinia virus, replication-defective adenoviral vectors and gutless adenovirus vectors (see, eg, US Patent No. 5,872,005, the entire contents of which are incorporated herein by reference). Methods for producing and isolating adeno-associated viruses (AAVs) suitable as vectors are known in the art (see, e.g., Grieger & Samulski, "Adeno-associated Virus as a Gene Therapy Vector: Vector Development, Production and Clinical Applications," Adv. Biochem. Engin/Biotechnol. 99: 119-145 (2005), Buning et al., "Recent Developments in Adeno-associated Virus Vector Technology," J. Gene Med. 10:717-733 (2008), Each of which is hereby incorporated by reference in its entirety).

編碼本文所述LukA和/或LukB蛋白或多肽的核酸分子通常與表達載體構建體中的啟動子序列、翻譯起始序列、3’非翻譯區序列、聚腺苷酸化序列和轉錄終止序列結合，以實現最大表達。適於驅動LukA和/或LukB蛋白或其多肽表達的啟動子序列包括但不限於延伸因子1-α(EF1a)啟動子、磷酸甘油酸激酶-1 (PGK)啟動子、巨細胞病毒立即早期基因啟動子(CMV)、嵌合肝特異性啟動子(LSP)、巨細胞病毒增強子/雞β-肌動蛋白啟動子(CAG)、四環素應答啟動子(TRE)、甲狀腺素運載蛋白啟動子(TTR)、猿猴病毒40啟動子(SV40)和CK6啟動子。本領域已知的適於驅動宿主細胞中基因表達的其他啟動子也適於包含在本文公開的表達構建體中。The nucleic acid molecule encoding the LukA and/or LukB protein or polypeptide described herein is usually combined with a promoter sequence, a translation initiation sequence, a 3' untranslated region sequence, a polyadenylation sequence and a transcription termination sequence in an expression vector construct, for maximum expression. Promoter sequences suitable for driving expression of LukA and/or LukB proteins or polypeptides thereof include, but are not limited to, elongation factor 1-alpha (EF1a) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus immediate early gene promoter (CMV), chimeric liver-specific promoter (LSP), cytomegalovirus enhancer/chicken β-actin promoter (CAG), tetracycline-responsive promoter (TRE), transthyretin promoter ( TTR), Simian virus 40 promoter (SV40) and CK6 promoter. Other promoters known in the art that are suitable for driving gene expression in a host cell are also suitable for inclusion in the expression constructs disclosed herein.

本發明的另一方面涉及包含載體的宿主細胞，所述載體包含編碼本文所述LukA和/或LukB多肽的多核苷酸。編碼本文所述LukA和LukB蛋白或多肽的表達構建體可以通過共轉染、串聯轉染或分別轉染到宿主細胞中。本文所述的LukA和LukB蛋白和多肽可以任選地由本領域所熟知的細胞系、混合細胞系、永生化細胞或永生化細胞的克隆群體產生(例如，參見Ausubel等人，ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001)；Sambrook等人，Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow和Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan等人，eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan等人，Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001)，其全部內容通過引用結合於此)。這種宿主細胞可以是真核細胞、細菌細胞、植物細胞或古細菌細胞。Another aspect of the invention pertains to host cells comprising a vector comprising a polynucleotide encoding a LukA and/or LukB polypeptide described herein. Expression constructs encoding the LukA and LukB proteins or polypeptides described herein can be transfected into host cells by co-transfection, tandem transfection, or separately. The LukA and LukB proteins and polypeptides described herein can optionally be produced from cell lines, mixed cell lines, immortalized cells, or clonal populations of immortalized cells well known in the art (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), the entire contents of which are hereby incorporated by reference). Such host cells may be eukaryotic, bacterial, plant or archaeal cells.

在任意的實施方案中，本文所述的LukA和/或LukB多肽在細菌細胞中產生。合適的細菌宿主細胞包括但不限於埃希氏菌宿主細胞、假單胞菌宿主細胞、葡萄球菌宿主細胞、鏈黴菌宿主細胞、分枝杆菌宿主細胞和芽孢杆菌宿主細胞。在任意的實施方案中，宿主細胞是大腸杆菌宿主細胞。在任意的實施方案中，宿主細胞是金黃色葡萄球菌宿主細胞。In any of the embodiments, the LukA and/or LukB polypeptides described herein are produced in bacterial cells. Suitable bacterial host cells include, but are not limited to, Escherichia host cells, Pseudomonas host cells, Staphylococcus host cells, Streptomyces host cells, Mycobacterium host cells, and Bacillus host cells. In any of the embodiments, the host cell is an E. coli host cell. In any of the embodiments, the host cell is a S. aureus host cell.

在任意的實施方案中，本文所述的LukA和/或LukB多肽在真核細胞中產生。示例性真核細胞可以來自哺乳動物、昆蟲、鳥類或其他動物。哺乳動物真核細胞包括永生化細胞系如雜交瘤或骨髓瘤細胞系如SP2/0(美國典型培養物保藏中心(ATCC)，弗吉尼亞州馬納薩斯，CRL-1581)，NSO(歐洲細胞培養物保藏中心(ECACC)，英國威爾特郡索爾茲伯裏市，UK, ECACC No. 85110503)，FO (ATCC CRL-1646)和Ag653 (ATCC CRL-1580)鼠細胞系。示例性的人骨髓瘤細胞系是U266 (ATTC CRL-TIB-196)。其他有用的細胞系包括來自中國倉鼠卵巢(CHO)細胞的細胞系，如CHO-K1SV (Lonza Biologics，Walkersville，Md .)、CHO-K1 (ATCC CRL-61)或DG44。In any of the embodiments, the LukA and/or LukB polypeptides described herein are produced in eukaryotic cells. Exemplary eukaryotic cells can be from mammals, insects, birds or other animals. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NSO (European Cell Culture Animal Collection Center (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, Md.), CHO-K1 (ATCC CRL-61 ) or DG44.

本文所述的LukA和LukB多肽可以使用上述分離的多核苷酸、載體和宿主細胞通過多種技術中的任何一種來制備。通常，蛋白質是通過標準克隆和細胞培養技術產生的，這些技術通常用於制備重組表達載體、轉染宿主細胞、選擇轉化體、培養宿主細胞以及從培養基中回收蛋白質或多肽。轉染宿主細胞可以使用多種常用於將外源性DNA導入原核或真核宿主細胞的技術來進行，例如通過電穿孔、磷酸鈣沉澱、DEAE-葡聚糖轉染等。The LukA and LukB polypeptides described herein can be produced by any of a variety of techniques using the isolated polynucleotides, vectors and host cells described above. Typically, proteins are produced by standard cloning and cell culture techniques commonly used to prepare recombinant expression vectors, transfect host cells, select for transformants, grow host cells, and recover the protein or polypeptide from the culture medium. Transfection of host cells can be performed using various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as by electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

本文所述的LukA和/或LukB多肽可以通過諸如糖基化、異構化、去糖基化等過程進行翻譯後修飾，或者通過非天然發生的共價修飾(諸如添加聚乙二醇(PEG)部分(聚乙二醇化)和脂質化等)進行翻譯後修飾。這種修飾可以發生在體內或體外。The LukA and/or LukB polypeptides described herein may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or by non-naturally occurring covalent modifications such as the addition of polyethylene glycol (PEG ) part (pegylation) and lipidation, etc.) are post-translationally modified. Such modifications can occur in vivo or in vitro.

在任意的實施方案中，本文所述的LukA和LukB多核苷酸和/或多肽優選為“分離的”多核苷酸和/或多肽。當用於描述本文公開的多核苷酸和/或多肽時，“分離的”意味著多核苷酸和/或多肽已經從其生產環境的組分中鑒定、分離和/或回收。優選地，分離的多核苷酸和/或多肽不與來自其生產環境的其他組分結合。其生產環境中的汙染物組分，例如由重組轉染細胞產生的汙染物成分，是通常會幹擾藥物使用的物質，可能包括酶、激素和其他蛋白質或非蛋白質溶質。通過已知方法從重組細胞培養物中回收和純化多核苷酸和/或多肽，包括但不限於蛋白A純化、硫酸銨或乙醇沉澱、酸提取、陰離子或陽離子交換色譜、磷酸纖維素色譜、疏水相互作用色譜、親和色譜、羥基磷灰石色譜和凝集素色譜。高效液相色譜法（“HPLC”）也可用於純化。In any of the embodiments, the LukA and LukB polynucleotides and/or polypeptides described herein are preferably "isolated" polynucleotides and/or polypeptides. "Isolated" when used to describe the polynucleotides and/or polypeptides disclosed herein means that the polynucleotides and/or polypeptides have been identified, separated and/or recovered from components of the environment in which they were produced. Preferably, an isolated polynucleotide and/or polypeptide is free from association with other components from the environment in which it was produced. Contaminant components in their production environment, such as those produced by recombinantly transfected cells, are substances that typically interfere with drug use and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Polynucleotides and/or polypeptides are recovered and purified from recombinant cell culture by known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic Interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

金黃色葡萄球菌疫苗組合物Staphylococcus aureus vaccine composition

本發明的另一方面涉及金黃色葡萄球菌疫苗組合物。在任意的實施方案中，所述金黃色葡萄球菌疫苗組合物包含本文所述的任何一種或多種LukA變異多肽，或編碼本文所述LukA變異多肽的一種或多種核酸分子。特別的，所述疫苗組合物的LukA變異多肽，如本文鑒定和描述的，在一個或多個氨基酸殘基的任何一個上包含氨基酸殘基***、取代和/或缺失。在任意的實施方案中，所述疫苗組合物的LukA變體包含SEQ ID NO:25的變體或SEQ ID NO:1、2或26-38中任何一個的變體。在任意的實施方案中，所述疫苗組合物的LukA變體包含SEQ ID NO:1 (CC8)的變體。示例性的CC8LukA變體包括但不限於SEQ ID NO:3、5、7、9和13的LukA變體。在任意的實施方案中，所述疫苗組合物的LukA變體包含SEQ ID NO:2 (CC45)的變體。示例性的CC45 LukA變體包括但不限於SEQ ID NOs: 4、6、8、10、11、12和14的LukA變體。Another aspect of the invention relates to S. aureus vaccine compositions. In any embodiment, the Staphylococcus aureus vaccine composition comprises any one or more variant LukA polypeptides described herein, or one or more nucleic acid molecules encoding the variant LukA polypeptides described herein. In particular, the LukA variant polypeptide of the vaccine composition, as identified and described herein, comprises an amino acid residue insertion, substitution and/or deletion at any one of one or more amino acid residues. In any of the embodiments, the LukA variant of the vaccine composition comprises a variant of SEQ ID NO: 25 or a variant of any one of SEQ ID NOs: 1, 2 or 26-38. In any of the embodiments, the LukA variant of the vaccine composition comprises a variant of SEQ ID NO: 1 (CC8). Exemplary CC8 LukA variants include, but are not limited to, the LukA variants of SEQ ID NO: 3, 5, 7, 9 and 13. In any of the embodiments, the LukA variant of the vaccine composition comprises a variant of SEQ ID NO: 2 (CC45). Exemplary CC45 LukA variants include, but are not limited to, the LukA variants of SEQ ID NOs: 4, 6, 8, 10, 11, 12, and 14.

在任意的實施方案中，本文公開的金黃色葡萄球菌疫苗組合物包含具有SEQ ID NO:3的氨基酸序列的CC8LukA變體。In any of the embodiments, the S. aureus vaccine composition disclosed herein comprises a CC8LukA variant having the amino acid sequence of SEQ ID NO:3.

在任意的實施方案中，本文公開的金黃色葡萄球菌疫苗組合物包含具有SEQ ID NO:7的氨基酸序列的CC8 LukA變體。In any of the embodiments, the S. aureus vaccine composition disclosed herein comprises a CC8 LukA variant having the amino acid sequence of SEQ ID NO:7.

在任意的實施方案中，本文公開的金黃色葡萄球菌疫苗組合物包含具有SEQ ID NO:8的氨基酸序列的CC45 LukA變體。In any of the embodiments, the S. aureus vaccine composition disclosed herein comprises a CC45 LukA variant having the amino acid sequence of SEQ ID NO:8.

在任意的實施方案中，本文公開的金黃色葡萄球菌疫苗組合物包含本文所述的任何一種或多種變異LukB蛋白或多肽，或編碼本文所述的LukB變體蛋白或多肽的一種或多種核酸分子。特別的，所述疫苗組合物的LukB變異多肽，如本文鑒定和描述的，在一個或多個氨基酸殘基的任何一個上包含氨基酸殘基***、取代和/或缺失。在任意的實施方案中，所述疫苗組合物的LukB變體包含SEQ ID NO:39的變體或SEQ ID NO:15、16或40-51中任何一個的變體。在任意的實施方案中，所述疫苗組合物的LukB變體包含SEQ ID NO:15 (CC8)的變體。示例性的CC8 LukB變體包括但不限於SEQ ID NO:17、19和21的LukB變體。在任意的實施方案中，所述疫苗組合物的LukB變體包含SEQ ID NO:16 (CC45)的變體。示例性的CC45 LukB變體包括但不限於SEQ ID NO:18、20和21的LukB變體。In any embodiment, the Staphylococcus aureus vaccine composition disclosed herein comprises any one or more variant LukB proteins or polypeptides described herein, or one or more nucleic acid molecules encoding the LukB variant proteins or polypeptides described herein . In particular, the LukB variant polypeptides of the vaccine composition, as identified and described herein, comprise amino acid residue insertions, substitutions and/or deletions at any of one or more amino acid residues. In any of the embodiments, the LukB variant of the vaccine composition comprises a variant of SEQ ID NO: 39 or a variant of any one of SEQ ID NOs: 15, 16, or 40-51. In any of the embodiments, the LukB variant of the vaccine composition comprises a variant of SEQ ID NO: 15 (CC8). Exemplary CC8 LukB variants include, but are not limited to, the LukB variants of SEQ ID NO: 17, 19 and 21. In any of the embodiments, the LukB variant of the vaccine composition comprises a variant of SEQ ID NO: 16 (CC45). Exemplary CC45 LukB variants include, but are not limited to, the LukB variants of SEQ ID NO: 18, 20, and 21.

在任意的實施方案中，本文公開的疫苗組合物同時包含LukA和LukB蛋白。因此，在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:1的CC8 LukA變體，同時包含SEQ ID NO:15的CC8 LukB非變體序列或具有與SEQ ID NO:15至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的變體序列。在任意的實施方案中，CC8 LukB序列變體序列包含選自SEQ ID NO:17、19和21的氨基酸序列。例如，在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:3的CC8 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB具有85%或更多序列同一性的變體，例如選自SEQ ID NO:17、19和21的CC8 LukB變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:5的CC8 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的CC8 LukB變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:7的CC8 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的CC8 LukB變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:9的CC8 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的CC8 LukB變體序列。In any of the embodiments, the vaccine compositions disclosed herein comprise both LukA and LukB proteins. Therefore, in any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO: 1, simultaneously comprising the CC8 LukB non-variant sequence of SEQ ID NO: 15 or having at least 85% of SEQ ID NO: 15 Variant sequences with %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity . In any of the embodiments, the CC8 LukB sequence variant sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 19 and 21. For example, in any of the embodiments, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO: 3 and at the same time comprises the CC8 LukB sequence of SEQ ID NO: 15 or is 85% identical to the CC8 LukB of SEQ ID NO: 15 or more sequence identity variants, for example CC8 LukB variant sequences selected from SEQ ID NO:17, 19 and 21. In any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO: 5, and simultaneously comprises the CC8 LukB sequence of SEQ ID NO: 15 or has more than 85% sequence with the CC8 LukB of SEQ ID NO: 15 Variants of identity, eg CC8 LukB variant sequences selected from SEQ ID NO:17, 19 and 21. In any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO: 7, while comprising the CC8 LukB sequence of SEQ ID NO: 15 or having more than 85% sequence with the CC8 LukB of SEQ ID NO: 15 Variants of identity, eg CC8 LukB variant sequences selected from SEQ ID NO:17, 19 and 21. In any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO: 9, while comprising the CC8 LukB sequence of SEQ ID NO: 15 or having more than 85% sequence with the CC8 LukB of SEQ ID NO: 15 Variants of identity, eg CC8 LukB variant sequences selected from SEQ ID NO:17, 19 and 21.

在任意的實施方案中，所述疫苗組合物包含具有SEQ ID NO:3氨基酸序列的CC8 LukA變體和具有SEQ ID NO:15氨基酸序列的CC8 LukB變體。In any of the embodiments, the vaccine composition comprises a CC8 LukA variant having the amino acid sequence of SEQ ID NO:3 and a CC8 LukB variant having the amino acid sequence of SEQ ID NO:15.

在任意的實施方案中，所述疫苗組合物包含具有SEQ ID NO:3氨基酸序列的CC8 LukA變體和具有SEQ ID NO:17氨基酸序列的CC8 LukB變異體。In any embodiment, the vaccine composition comprises a CC8 LukA variant having the amino acid sequence of SEQ ID NO:3 and a CC8 LukB variant having the amino acid sequence of SEQ ID NO:17.

在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:1的CC8 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或具有與SEQ ID NO:16至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的變體序列。在任意的實施方案中，CC45 LukB變體序列包含選自SEQ ID NO:18、20和22的氨基酸序列。例如，在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:3的CC8 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:5的CC8 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:7的CC8 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:9的CC8 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體序列。In any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO:1, and at the same time comprises the CC45 LukB sequence of SEQ ID NO:16 or has at least 85%, 86%, Variant sequences with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In any of the embodiments, the CC45 LukB variant sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 20 and 22. For example, in any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO: 3, and simultaneously comprises the CC45 LukB sequence of SEQ ID NO: 16 or has a sequence greater than 85 with the CC45 LukB of SEQ ID NO: 16. Variants with % sequence identity, for example CC45 LukB variant sequences selected from SEQ ID NO: 18, 20 and 22. In any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO:5, and simultaneously comprises the CC45 LukB sequence of SEQ ID NO:16 or has more than 85% sequence with the CC45 LukB of SEQ ID NO:16 Variants of identity, eg CC45 LukB variant sequences selected from SEQ ID NO: 18, 20 and 22. In any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO: 7, while comprising the CC45 LukB sequence of SEQ ID NO: 16 or having more than 85% sequence with the CC45 LukB of SEQ ID NO: 16 Variants of identity, eg CC45 LukB variant sequences selected from SEQ ID NO: 18, 20 and 22. In any embodiment, the vaccine composition comprises the CC8 LukA variant of SEQ ID NO: 9, while comprising the CC45 LukB sequence of SEQ ID NO: 16 or having more than 85% sequence with the CC45 LukB of SEQ ID NO: 16 Variants of identity, eg CC45 LukB variant sequences selected from SEQ ID NO: 18, 20 and 22.

在任意的實施方案中，所述疫苗組合物包含具有SEQ ID NO:5氨基酸序列的CC8 LukA變體和具有SEQ ID NO:16氨基酸序列的CC45 LukB變體。In any of the embodiments, the vaccine composition comprises a CC8 LukA variant having the amino acid sequence of SEQ ID NO:5 and a CC45 LukB variant having the amino acid sequence of SEQ ID NO:16.

在任意的實施方案中，所述疫苗組合物包含具有SEQ ID NO:5氨基酸序列的CC8 LukA變體和具有SEQ ID NO:22氨基酸序列的CC45 LukB變體。In any of the embodiments, the vaccine composition comprises a CC8 LukA variant having the amino acid sequence of SEQ ID NO:5 and a CC45 LukB variant having the amino acid sequence of SEQ ID NO:22.

在任意的實施方案中，所述疫苗組合物包含具有SEQ ID NO:5氨基酸序列的CC8 LukA變體和具有SEQ ID NO:18氨基酸序列的CC45 LukB變體。In any of the embodiments, the vaccine composition comprises a CC8 LukA variant having the amino acid sequence of SEQ ID NO:5 and a CC45 LukB variant having the amino acid sequence of SEQ ID NO:18.

在任意的實施方案中，所述疫苗組合物包含具有SEQ ID NO:5氨基酸序列的CC8 LukA變體和具有SEQ ID NO:20氨基酸序列的CC45 LukB變體。In any of the embodiments, the vaccine composition comprises a CC8 LukA variant having the amino acid sequence of SEQ ID NO:5 and a CC45 LukB variant having the amino acid sequence of SEQ ID NO:20.

在任意的實施方案中，所述疫苗組合物包含具有SEQ ID NO:3氨基酸序列的變異LukA蛋白和具有SEQ ID NO:18氨基酸序列的LukB蛋白。In any embodiment, the vaccine composition comprises a variant LukA protein having the amino acid sequence of SEQ ID NO:3 and a LukB protein having the amino acid sequence of SEQ ID NO:18.

在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:2的CC45 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或具有與SEQ ID NO:16至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的變體序列。在任意的實施方案中，CC45 LukB變體序列包含選自SEQ ID NO:18、20和22的氨基酸序列。例如，在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:4的CC45 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:6的CC45 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:8的CC45 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:10的CC45 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:11的CC45 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:12的CC45 LukA變體，同時包含SEQ ID NO:16的CC45 LukB序列或與SEQ ID NO:16的CC45 LukB具有大於 85%序列同一性的變體，例如選自SEQ ID NO:18、20和22的CC45 LukB變體。In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 2, and at the same time comprises the CC45 LukB sequence of SEQ ID NO: 16 or has at least 85%, 86%, Variant sequences with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In any of the embodiments, the CC45 LukB variant sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 20 and 22. For example, in any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 4, and at the same time comprises the CC45 LukB sequence of SEQ ID NO: 16 or has a sequence greater than 85 with the CC45 LukB of SEQ ID NO: 16. Variants with % sequence identity, for example CC45 LukB variant sequences selected from SEQ ID NO: 18, 20 and 22. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 6, while comprising the CC45 LukB sequence of SEQ ID NO: 16 or having more than 85% sequence with the CC45 LukB of SEQ ID NO: 16 Variants of identity, eg CC45 LukB variant sequences selected from SEQ ID NO: 18, 20 and 22. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 8, while comprising the CC45 LukB sequence of SEQ ID NO: 16 or having more than 85% sequence with the CC45 LukB of SEQ ID NO: 16 Variants of identity, eg CC45 LukB variants selected from SEQ ID NO: 18, 20 and 22. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 10, while comprising the CC45 LukB sequence of SEQ ID NO: 16 or having more than 85% sequence with the CC45 LukB of SEQ ID NO: 16 Variants of identity, eg CC45 LukB variants selected from SEQ ID NO: 18, 20 and 22. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 11, while comprising the CC45 LukB sequence of SEQ ID NO: 16 or having more than 85% sequence with the CC45 LukB of SEQ ID NO: 16 Variants of identity, eg CC45 LukB variants selected from SEQ ID NO: 18, 20 and 22. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 12, while comprising the CC45 LukB sequence of SEQ ID NO: 16 or having more than 85% sequence with the CC45 LukB of SEQ ID NO: 16 Variants of identity, eg CC45 LukB variants selected from SEQ ID NO: 18, 20 and 22.

在一個實施方案中，所述疫苗組合物包含具有SEQ ID NO:4的氨基酸序列的CC45 LukA變體與具有SEQ ID NO:16的氨基酸序列的CC45 LukB的組合。In one embodiment, the vaccine composition comprises a CC45 LukA variant having the amino acid sequence of SEQ ID NO:4 in combination with CC45 LukB having the amino acid sequence of SEQ ID NO:16.

在一個實施方案中，所述疫苗組合物包含具有SEQ ID NO:11的氨基酸序列的CC45 LukA變體與具有SEQ ID NO:16的氨基酸序列的CC45 LukB的組合。In one embodiment, the vaccine composition comprises a CC45 LukA variant having the amino acid sequence of SEQ ID NO: 11 in combination with CC45 LukB having the amino acid sequence of SEQ ID NO: 16.

在一個實施方案中，所述疫苗組合物包含具有SEQ ID NO:12的氨基酸序列的CC45 LukA變體與具有SEQ ID NO:16的氨基酸序列的CC45 LukB的組合。In one embodiment, the vaccine composition comprises a CC45 LukA variant having the amino acid sequence of SEQ ID NO: 12 in combination with CC45 LukB having the amino acid sequence of SEQ ID NO: 16.

在一個實施方案中，所述疫苗組合物包含具有SEQ ID NO:8的氨基酸序列的CC45 LukA變體與具有SEQ ID NO:16的氨基酸序列的CC45 LukB的組合。In one embodiment, the vaccine composition comprises a CC45 LukA variant having the amino acid sequence of SEQ ID NO:8 in combination with CC45 LukB having the amino acid sequence of SEQ ID NO:16.

在一個實施方案中，所述疫苗組合物包含具有SEQ ID NO:4的氨基酸序列的CC45 LukA變體和具有SEQ ID NO:18的氨基酸序列的CC45 LukB變體的組合。In one embodiment, the vaccine composition comprises a combination of a CC45 LukA variant having the amino acid sequence of SEQ ID NO:4 and a CC45 LukB variant having the amino acid sequence of SEQ ID NO:18.

在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:2的CC45 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或具有與SEQ ID NO:15至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的變體序列。在任意的實施方案中，CC8 LukB變體序列包含選自SEQ ID NO:17、19和21的氨基酸序列。例如，在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:4的CC45 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB序列具有大於85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:6的CC45 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB序列具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:8的CC45 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB序列具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:9的CC45 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB序列具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:10的CC45 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB序列具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:11的CC45 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB序列具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:12的CC45 LukA變體，同時包含SEQ ID NO:15的CC8 LukB序列或與SEQ ID NO:15的CC8 LukB序列具有大於 85%序列同一性的變體，例如選自SEQ ID NO:17、19和21的變體序列。In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO:2, while comprising the CC8 LukB sequence of SEQ ID NO:15 or having at least 85%, 86%, Variant sequences with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In any of the embodiments, the CC8 LukB variant sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 19 and 21. For example, in any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 4, and simultaneously comprises the CC8 LukB sequence of SEQ ID NO: 15 or has a greater than or equal to the CC8 LukB sequence of SEQ ID NO: 15 A variant with 85% sequence identity, for example a variant sequence selected from SEQ ID NO: 17, 19 and 21. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO:6, and at the same time comprises the CC8 LukB sequence of SEQ ID NO:15 or has greater than 85% with the CC8 LukB sequence of SEQ ID NO:15 Variants of sequence identity, eg variant sequences selected from the group consisting of SEQ ID NO: 17, 19 and 21. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO:8, and simultaneously comprises the CC8 LukB sequence of SEQ ID NO:15 or has greater than 85% of the CC8 LukB sequence of SEQ ID NO:15 Variants of sequence identity, eg variant sequences selected from the group consisting of SEQ ID NO: 17, 19 and 21. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO:9, and simultaneously comprises the CC8 LukB sequence of SEQ ID NO:15 or has greater than 85% with the CC8 LukB sequence of SEQ ID NO:15 Variants of sequence identity, eg variant sequences selected from the group consisting of SEQ ID NO: 17, 19 and 21. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 10, and at the same time comprises the CC8 LukB sequence of SEQ ID NO: 15 or has greater than 85% of the CC8 LukB sequence of SEQ ID NO: 15 Variants of sequence identity, eg variant sequences selected from the group consisting of SEQ ID NO: 17, 19 and 21. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 11, and at the same time comprises the CC8 LukB sequence of SEQ ID NO: 15 or has greater than 85% with the CC8 LukB sequence of SEQ ID NO: 15 Variants of sequence identity, eg variant sequences selected from the group consisting of SEQ ID NO: 17, 19 and 21. In any embodiment, the vaccine composition comprises the CC45 LukA variant of SEQ ID NO: 12, and at the same time comprises the CC8 LukB sequence of SEQ ID NO: 15 or has greater than 85% of the CC8 LukB sequence of SEQ ID NO: 15 Variants of sequence identity, eg variant sequences selected from the group consisting of SEQ ID NO: 17, 19 and 21.

本發明的另一方面涉及金黃色葡萄球菌疫苗組合物，其包含本文所述的任何變異LukB變異多肽或編碼LukB變體的核酸分子。特別地，所述疫苗組合物的LukB變異多肽包含一個或多個本文所述的氨基酸殘基的***、取代和/或缺失。在任意的實施方案中，所述疫苗組合物的LukB變體包含SEQ ID NO:15 (CC8)的變體。示例性的CC8 LukB變體包括但不限於SEQ ID NO:17、19和21的LukB變體。在任意的實施方案中，所述疫苗組合物的LukB變體包含SEQ ID NO:16 (CC45)的變體。示例性的CC45 LukB變體包括但不限於SEQ ID NO:18、20和22的LukB變體。Another aspect of the invention relates to a S. aureus vaccine composition comprising any of the variant LukB variant polypeptides described herein or nucleic acid molecules encoding LukB variants. In particular, the LukB variant polypeptide of the vaccine composition comprises one or more insertions, substitutions and/or deletions of the amino acid residues described herein. In any of the embodiments, the LukB variant of the vaccine composition comprises a variant of SEQ ID NO: 15 (CC8). Exemplary CC8 LukB variants include, but are not limited to, the LukB variants of SEQ ID NO: 17, 19 and 21. In any of the embodiments, the LukB variant of the vaccine composition comprises a variant of SEQ ID NO: 16 (CC45). Exemplary CC45 LukB variants include, but are not limited to, the LukB variants of SEQ ID NO: 18, 20, and 22.

在任意的實施方案中，本文公開的疫苗組合物包含本文描述的LukB變異多肽和LukA蛋白或多肽。因此，在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:15的CC8 LukB變體，例如SEQ ID NO:17、19和21的變體，同時包含SEQ ID NO:1的CC8 LukA序列或具有與SEQ ID NO:1至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:15的CC8 LukB變體，例如SEQ ID NO:17、19和21的變體，同時包含SEQ ID NO:2的CC45 LukA序列或具有與SEQ ID NO:2至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的變體序列。In any of the embodiments, a vaccine composition disclosed herein comprises a LukB variant polypeptide and a LukA protein or polypeptide described herein. Thus, in any of the embodiments, the vaccine composition comprises a CC8 LukB variant of SEQ ID NO: 15, such as the variants of SEQ ID NO: 17, 19 and 21, together with the CC8 LukA of SEQ ID NO: 1 The sequence or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQ ID NO: 1 Variant sequences with % or 99% sequence identity. In any embodiment, the vaccine composition comprises a CC8 LukB variant of SEQ ID NO: 15, such as variants of SEQ ID NO: 17, 19 and 21, while comprising a CC45 LukA sequence of SEQ ID NO: 2 or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Variant sequences with 99% sequence identity.

在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:16的CC45 LukB變體，例如SEQ ID NO:18、20或22的變體，同時包含SEQ ID NO:1的CC8 LukA序列或具有與SEQ ID NO:1至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的變體序列。在任意的實施方案中，所述疫苗組合物包含SEQ ID NO:16的CC45 LukB變體，例如SEQ ID NO:18、20或22的變體，同時包含SEQ ID NO:2的CC45 LukA序列或具有與SEQ ID NO:2至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的變體序列。In any embodiment, the vaccine composition comprises a CC45 LukB variant of SEQ ID NO: 16, such as a variant of SEQ ID NO: 18, 20 or 22, while comprising a CC8 LukA sequence of SEQ ID NO: 1 or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Variant sequences with 99% sequence identity. In any embodiment, the vaccine composition comprises a CC45 LukB variant of SEQ ID NO: 16, such as a variant of SEQ ID NO: 18, 20 or 22, while comprising a CC45 LukA sequence of SEQ ID NO: 2 or have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Variant sequences with 99% sequence identity.

本發明的疫苗組合物是通過將本文所述的LukA和/或LukB多肽與藥學上可接受的載體和任選的藥學上可接受的賦形劑一起配制而制備的。藥學活性成分與藥學上可接受的載體的制備在本領域是已知的，例如Remington: The Science and Practice of Pharmacy (例如第21 版(2005)和任何更新的版本)。附加成分的非限制性例子包括:緩沖劑、稀釋劑、溶劑、張力調節劑、防腐劑、穩定劑和螯合劑。一種或多種藥學上可接受的載體可用於配制本發明的藥物組合物。The vaccine composition of the present invention is prepared by formulating the LukA and/or LukB polypeptides described herein together with a pharmaceutically acceptable carrier and optionally a pharmaceutically acceptable excipient. The preparation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, eg Remington: The Science and Practice of Pharmacy (eg 21st Edition (2005) and any later editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity adjusting agents, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers may be used to formulate the pharmaceutical compositions of the present invention.

如本文所用，術語“藥學上可接受的載體”和“藥學上可接受的賦形劑”(例如添加劑，如稀釋劑、免疫刺激劑、佐劑、抗氧化劑、防腐劑和增溶劑)以其所用的劑量和濃度施用組合物對受試者無毒。藥學上可接受的載體的例子包括水溶液，例如用磷酸鹽、檸檬酸鹽和另一種有機酸緩沖得到的水溶液。可用於本發明的藥學上可接受的賦形劑的代表性例子包括抗氧化劑，如抗壞血酸；低分子量(小於約10個殘基)多肽；蛋白質，如血清白蛋白、明膠或免疫球蛋白；親水聚合物，如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、穀氨醯胺、天冬醯胺、精氨酸或賴氨酸；單糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合劑，如乙二胺四乙酸；糖醇，如甘露醇或山梨醇；成鹽抗衡離子，如鈉；和/或非離子表面活性劑。As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives, and solubilizers) are used in conjunction with their The dosages and concentrations used to administer the composition are nontoxic to the subject. Examples of pharmaceutically acceptable carriers include aqueous solutions, eg, buffered with phosphate, citrate and another organic acid. Representative examples of pharmaceutically acceptable excipients that can be used in the invention include antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; Chelating agents, such as ethylenediaminetetraacetic acid; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants.

在任意的實施方案中，本文所述的疫苗組合物是液體制劑。液體制劑的優選實例是水性制劑，即包含水的制劑。液體制劑可以包括溶液、懸浮液、乳液、微乳液、凝膠等。水性制劑通常包含至少50% w/w的水，或至少60%、70%、75%、80%、85%、90%或至少95% w/w的水。In any of the embodiments, the vaccine compositions described herein are liquid formulations. A preferred example of a liquid formulation is an aqueous formulation, ie a formulation comprising water. Liquid formulations may include solutions, suspensions, emulsions, microemulsions, gels, and the like. Aqueous formulations typically comprise at least 50% w/w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w/w water.

疫苗組合物可以進一步包含一種或多種佐劑。如本文所用，術語“佐劑”是指當與本文所述的LukA和/或LukB多肽聯合給藥時，可以增加、增強和/或促進對多肽的免疫應答的化合物。然而，當佐劑化合物單獨給藥時，不會對上述多肽或編碼多肽的多核苷酸產生免疫應答。佐劑可以通過幾種機制增強免疫應答，包括例如淋巴細胞募集、刺激B和/或T細胞以及刺激抗原呈遞細胞。The vaccine composition may further comprise one or more adjuvants. As used herein, the term "adjuvant" refers to a compound that, when administered in combination with a LukA and/or LukB polypeptide described herein, increases, enhances and/or facilitates the immune response to the polypeptide. However, when the adjuvant compound is administered alone, it does not generate an immune response to the above-mentioned polypeptides or polynucleotides encoding the polypeptides. Adjuvants can enhance the immune response through several mechanisms including, for example, lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of antigen presenting cells.

本文所述的疫苗組合物包含LukA和/或LukB多肽和/或編碼該多肽的多核苷酸，所述疫苗組合物包含佐劑或與佐劑聯合施用。用於與本文所述疫苗組合物聯合施用的佐劑可以在施用疫苗組合物之前、同時或之後施用。The vaccine compositions described herein comprise LukA and/or LukB polypeptides and/or polynucleotides encoding the polypeptides, which comprise or are administered in combination with an adjuvant. Adjuvants for administration in conjunction with the vaccine compositions described herein may be administered prior to, simultaneously with, or after administration of the vaccine compositions.

合適的佐劑是本領域已知的，包括但不限於鞭毛蛋白、弗氏完全佐劑、弗氏不完全佐劑、氫氧化鋁、溶血卵磷脂、pluronic多元醇、聚陰離子、肽、油乳劑、二硝基酚、ISCOMATRIX和脂質體聚陽離子DNA顆粒。佐劑的其他例子包括，例如，專利US6,355,625中描述的β-葡聚糖(該專利在此全文引入作為參考)，或粒細胞集落刺激因子(GCSF)。Suitable adjuvants are known in the art and include, but are not limited to, flagellin, Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions , Dinitrophenol, ISCOMATRIX, and liposomal polycationic DNA particles. Other examples of adjuvants include, for example, beta-glucan as described in patent US 6,355,625 (which patent is hereby incorporated by reference in its entirety), or granulocyte colony stimulating factor (GCSF).

額外的示例性佐劑包括但不限於鋁鹽(明礬)(例如氫氧化鋁、磷酸鋁、硫酸鋁和氧化鋁，包括包含明礬或納米明礬制劑的納米顆粒)、磷酸鈣(例如，Masson JD等人，Expert Rev Vaccines 16: 289-299 (2017)，其在此全文引入作為參考)、單磷醯基脂質A (MPL)或3-脫氧-醯化單磷醯基脂質A (3D-MPL)(參見例如，GB2220211，EP0971739, EP1194166, US6491919，這些文獻在此全文引入作為參考)，AS01，AS02、AS03和AS04(例如參見EP1126876、記載了AS04的US7357936、EP0671948、EP0761231、記載了AS02的US5750110，其在此全文引入作為參考)、咪唑並吡啶化合物(參見WO2007/109812，其在此全文引入作為參考)、咪唑並喹喔啉化合物(參見WO2007/109813，其在此全文引入作為參考)、Δ-菊粉(例如Petrovsky N 和PD Cooper, Vaccine 33: 5920-5926 (2015)，其在此全文引入作為參考)、 STING-激活合成環狀二核苷酸(例如US20150056224，其在此全文引入作為參考)、卵磷脂和卡波姆均聚物的組合(例如US6,676,958，其在此全文引入作為參考)，和皂苷，例如Quil A和QS21(例如參見Zhu D和W Tuo，2016, Nat Prod Chem Res 3: e113 (doi:10.4172/2329-6836.1000e113)，其在此全文引入作為參考)，任選地與QS7(參見例如Kensil等人，in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US 5,057,540，其在此全文引入作為參考)組合。在任意的實施方案中，所述佐劑是弗氏佐劑(完全或不完全)。在任意的實施方案中，所述佐劑包含Quil-A，例如可從Brenntag(現稱Croda)或Invivogen購買得到的Quil-A。Quil-A含有來自於Quillaja saponaria Molina tree的皂苷的水提成分。這些皂苷屬於三萜皂苷類，具有共同的三萜骨架結構。已知皂苷可誘導對T依賴和T非依賴抗原的強佐劑反應，以及強細胞毒性CD8+淋巴細胞反應，並增強對粘膜抗原的反應。因此，在任意的實施方案中，所述佐劑包含皂苷。在任意的實施方案中，所述佐劑包含QS-21。Additional exemplary adjuvants include, but are not limited to, aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles comprising alum or nanoalum formulations), calcium phosphate (e.g., Masson JD et al. Human, Expert Rev Vaccines 16: 289-299 (2017), which is hereby incorporated by reference in its entirety), monophosphoryl lipid A (MPL) or 3-deoxy-acylated monophosphoryl lipid A (3D-MPL) (see for example GB2220211, EP0971739, EP1194166, US6491919, which are hereby incorporated by reference in their entirety), AS01, AS02, AS03 and AS04 (see for example EP1126876, US7357936 which describes AS04, EP0671948, EP0761231, US5750 which describes AS02 110, which is hereby incorporated by reference in its entirety), imidazopyridine compounds (see WO2007/109812, which is hereby incorporated by reference in its entirety), imidazoquinoxaline compounds (see WO2007/109813, which is hereby incorporated by reference in its entirety), Δ - Inulin (e.g. Petrovsky N and PD Cooper, Vaccine 33: 5920-5926 (2015), which is hereby incorporated by reference in its entirety), STING-activated synthetic cyclic dinucleotides (e.g. US20150056224, which is hereby incorporated in its entirety as reference), lecithin and carbomer homopolymer combinations (for example US6,676,958, which is hereby incorporated by reference in its entirety), and saponins such as Quil A and QS21 (for example see Zhu D and W Tuo, 2016, Nat Prod Chem Res 3: e113 (doi:10.4172/2329-6836.1000e113), which is hereby incorporated by reference in its entirety), optionally with QS7 (see, e.g., Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US 5,057,540, which is hereby incorporated by reference in its entirety) in combination. In any embodiment, the adjuvant is Freund's adjuvant (complete or incomplete). In any of the embodiments, the adjuvant comprises Quil-A, such as Quil-A commercially available from Brenntag (now known as Croda) or Invivogen. Quil-A contains an aqueous extract of saponins from the Quillaja saponaria Molina tree. These saponins belong to triterpene saponins and have a common triterpene skeleton structure. Saponins are known to induce strong adjuvant responses to T-dependent and T-independent antigens, as well as strong cytotoxic CD8+ lymphocyte responses and enhance responses to mucosal antigens. Thus, in any of the embodiments, the adjuvant comprises a saponin. In any of the embodiments, the adjuvant comprises QS-21.

在任意的實施方案中，所述皂苷與膽固醇和磷脂結合，形成免疫刺激複合物(ISCOM)，其可以啟動抗體介導和細胞介導的針對不同來源的廣泛抗原的免疫反應。在某些實施方案中，所述佐劑是AS01，例如AS01B。AS01是含有MPL(3-O-去十二烷基-4’-單磷醯基脂質A)、QS21(Quillaja saponaria Molina，餾分21)和脂質體的佐劑系統。在某些實施方案中，AS01是商業上可購買得到的，或者可以如WO 96/33739中所述制備，該文獻通過引用整體結合於此。某些助劑包括乳液，乳液是兩種不混溶的流體，例如油和水的混合物，其中一種以小液滴形式懸浮在另一種流體中，並通過表面活性劑穩定。水包油乳液具有形成連續相的水，圍繞小油滴，而油包水乳液具有形成連續相的油。某些水包油乳液包含角鯊烯(一種可代謝的油)。某些助劑包括嵌段共聚物，它是當兩種單體聚集在一起並形成重複單元的嵌段時形成的共聚物。包含嵌段共聚物、角鯊烯和微粒穩定劑的油包水乳液的一個例子是TiterMax®，其可以從Sigma-Aldrich公司購買得到。In any of the embodiments, the saponins combine with cholesterol and phospholipids to form immunostimulatory complexes (ISCOMs) that can initiate antibody-mediated and cell-mediated immune responses against a wide range of antigens of different origin. In certain embodiments, the adjuvant is AS01, such as AS01B. AS01 is an adjuvant system containing MPL (3-O-desdodecyl-4'-monophosphoryl lipid A), QS21 (Quillaja saponaria Molina, fraction 21 ) and liposomes. In certain embodiments, AS01 is commercially available or can be prepared as described in WO 96/33739, which is hereby incorporated by reference in its entirety. Certain adjuvants include emulsions, which are mixtures of two immiscible fluids, such as oil and water, one of which is suspended in the other in the form of small droplets, stabilized by surfactants. Oil-in-water emulsions have water forming the continuous phase, surrounding small oil droplets, while water-in-oil emulsions have oil forming the continuous phase. Certain oil-in-water emulsions contain squalene, a metabolizable oil. Certain additives include block copolymers, which are copolymers formed when two monomers are brought together and form blocks of repeating units. An example of a water-in-oil emulsion comprising a block copolymer, squalene and a particulate stabilizer is TiterMax®, commercially available from Sigma-Aldrich Corporation.

任選地，乳劑可以與其他免疫刺激成分如TLR4激動劑結合或包含其他的免疫刺激成分。適用於本文公開的組合物中的佐劑組合的合適但非限制性的例子包括水包油乳液(如角鯊烯或花生油)、MF59(例如參見EP0399843、US 6299884、US6451325，其全部內容通過引用結合於此)和AS03，任選與免疫刺激劑如單磷醯脂質A和/或QS21如AS02(參見Stoute等人，N. Engl. J. Med336: 86-91 (1997)s，其全部內容通過引用結合於此)進行組合。佐劑的其他示例是含有免疫刺激劑如MPL和QS21的脂質體，如在AS01E和AS01B中(例如，參見US2011/0206758，其通過引用整體結合於此)。佐劑的其他例子是CpG和咪唑並喹啉(如咪喹莫特和R848)(例如，參見Reed G等人.，Nature Med，19: 1597-1608 (2013)，其全部內容通過引用結合於此)。在本發明所述的任何實施方案中，所述佐劑是Th1佐劑。Optionally, the emulsion may be combined with or contain other immunostimulatory ingredients, such as TLR4 agonists. Suitable but non-limiting examples of adjuvant combinations suitable for use in the compositions disclosed herein include oil-in-water emulsions such as squalene or peanut oil, MF59 (see for example EP0399843, US 6299884, US6451325, the entire contents of which are incorporated by reference incorporated herein) and AS03, optionally with immunostimulants such as monophosphoryl lipid A and/or QS21 such as AS02 (see Stoute et al., N. Engl. J. Med336: 86-91 (1997)s, its entirety incorporated herein by reference). Other examples of adjuvants are liposomes containing immunostimulants such as MPL and QS21, as in AS01E and AS01B (see eg US2011/0206758, which is hereby incorporated by reference in its entirety). Other examples of adjuvants are CpG and imidazoquinolines (such as imiquimod and R848) (see, for example, Reed G et al., Nature Med, 19: 1597-1608 (2013), the entire contents of which are incorporated by reference at this). In any of the embodiments described herein, the adjuvant is a Th1 adjuvant.

在任意的實施方案中，本文公開的疫苗組合物的佐劑包含單獨的toll樣受體4 (TLR4)激動劑或其與另一種佐劑的組合。TLR4激動劑在本領域中是眾所周知的，例如參見Ireton GC和SG Reed，Expert Rev ventures 12:793-807(2013)，其全部內容通過引用結合於此。在任意的實施方案中，佐劑是包含脂質A或其類似物或衍生物的TLR4激動劑。In any of the embodiments, the adjuvant of the vaccine compositions disclosed herein comprises a toll-like receptor 4 (TLR4) agonist alone or in combination with another adjuvant. TLR4 agonists are well known in the art, see eg Ireton GC and SG Reed, Expert Rev ventures 12:793-807 (2013), the entire contents of which are hereby incorporated by reference. In any of the embodiments, the adjuvant is a TLR4 agonist comprising lipid A or an analog or derivative thereof.

在任意的實施方案中，所述疫苗組合物的佐劑包含脂質A或脂質A類似物或衍生物。如本文所用，術語“脂質A”是指脂多糖分子(LPS)的疏水脂質部分，其包含氨基葡萄糖，並通過酮苷鍵與脂多糖分子內核中的酮基-脫氧辛基酮糖酸連接，該酮苷鍵將脂多糖分子錨定在革蘭氏陰性菌外膜的外小葉中。本文所用的脂質A包括天然存在的脂質A、其混合物、類似物、衍生物和前體。該術語包括單糖，例如脂質A的前體，稱為脂質X；二糖脂質A；七醯基脂質A；六醯基脂質A；五醯基脂質A；四醯基脂質A，例如脂質A的四醯基前體，稱為脂質IVA；去磷酸化脂質A；單磷醯基脂質A；二磷醯基脂質A，如來自大腸杆菌和球形紅杆菌的脂質A。幾種免疫激活脂質A結構含有6條醯基鏈。直接連接到葡糖胺糖上的四個初級(primary)醯基鏈是3-羥基醯基鏈，長度通常在10到16個碳之間。兩個額外的醯基鏈通常連接在初級醯基鏈的3-羥基上。例如，大腸杆菌脂質A通常具有四個連接到糖上的C14 3-羥基醯基鏈，以及一個C12和一個C14分別連接到初級醯基鏈的2’和3’位的3-羥基上。In any of the embodiments, the adjuvant of the vaccine composition comprises lipid A or a lipid A analog or derivative. As used herein, the term "lipid A" refers to the hydrophobic lipid portion of a lipopolysaccharide molecule (LPS), which contains glucosamine and is linked to keto-deoxyoctylgluconic acid in the inner core of the lipopolysaccharide molecule by a ketoglycosidic bond, This ketoglycosidic bond anchors the lipopolysaccharide molecule in the outer leaflet of the outer membrane of Gram-negative bacteria. Lipid A as used herein includes naturally occurring lipid A, mixtures, analogs, derivatives and precursors thereof. The term includes monosaccharides such as the precursor of lipid A, known as lipid X; disaccharide lipid A; heptayl lipid A; hexacyl lipid A; pentaacyl lipid A; Dephosphorylated lipid A; monophosphoryl lipid A; diphosphoryl lipid A, such as lipid A from Escherichia coli and Rhodobacter sphaericus. Several immunostimulatory lipid A structures contain 6 acyl chains. The four primary acyl chains directly attached to the glucosamine sugar are 3-hydroxyacyl chains, typically between 10 and 16 carbons in length. Two additional acyl chains are usually attached to the 3-hydroxyl group of the primary acyl chain. For example, E. coli lipid A typically has four C14 3-hydroxyacyl chains attached to the sugar, and one C12 and one C14 attached to the 3-hydroxyls at the 2' and 3' positions of the primary acyl chain, respectively.

如本文所用，術語“脂質A類似物或衍生物”是指具有類似於脂質A的結構和免疫活性，但不一定天然存在的分子。脂質A類似物或衍生物可以被修飾以被縮短或縮合，和/或使它們的葡糖胺殘基被另一個胺糖殘基(例如半乳糖胺殘基)取代，以在還原端含有2-脫氧-2-氨基葡萄糖酸鹽以代替葡糖胺-1-磷酸鹽，在4’位帶有半乳糖醛酸部分而不是磷酸鹽。脂質A類似物或衍生物可由從細菌中分離的脂質A制備，例如通過化學衍生，或化學合成，例如通過首先確定優選脂質A的結構並合成其類似物或衍生物。脂質A類似物或衍生物也可用作TLR4激動劑佐劑(參見，例如，Gregg KA等人，MBio 8, eDD492-17, doi: 10.1128/mBio.00492-17 (2017)，其全部內容通過引用結合於此)。As used herein, the term "lipid A analog or derivative" refers to a molecule that has a structure and immunological activity similar to lipid A, but does not necessarily occur naturally. Lipid A analogs or derivatives can be modified to be shortened or condensed, and/or have their glucosamine residue replaced by another amine sugar residue (e.g., a galactosamine residue) to contain 2 at the reducing end. - Deoxy-2-glucosamine in place of glucosamine-1-phosphate, with a galacturonic acid moiety at the 4' position instead of a phosphate. Lipid A analogs or derivatives can be prepared from lipid A isolated from bacteria, eg, by chemical derivatization, or chemically synthesized, eg, by first determining the structure of a preferred lipid A and synthesizing an analog or derivative thereof. Lipid A analogs or derivatives can also be used as TLR4 agonist adjuvants (see, e.g., Gregg KA et al., MBio 8, eDD492-17, doi: 10.1128/mBio.00492-17 (2017), accessed in its entirety incorporated herein by reference).

MPL和3D-MPL是脂質A類似物或衍生物，已被修飾以減輕脂質A的毒性。脂質A、MPL和3D-MPL具有糖主鏈，有長脂肪酸鏈連接在糖主鏈上，其中所述主鏈包含糖苷鍵中的兩個6碳糖和4位的磷醯基部分。典型地，五到八個長鏈脂肪酸(通常12-14個碳原子)連接到糖主鏈上。由於天然來源的衍生，MPL或3D-MPL可以以多種脂肪酸取代模式(例如七醯基、六醯基、五醯基等，其具有可變的脂肪酸長度)的複合物或混合物的形式存在。對於本文所述的一些其他脂質A類似物或衍生物也是如此，然而合成脂質A變異體也可以被定義和均質化。US4,436,727中記載了MPL及其批量生產，該專利在此全文引入作為參考。US4,912,094B1中描述了3D-MPL(該專利在此全文引入作為參考)，並且與MPL的不同之處在於選擇性去除了3-羥基肉豆蔻醯基殘基，該殘基在3位與還原端的氨基葡萄糖酯連接。適於本文所述疫苗組合物中的脂質A(類似物、衍生物)的例子包括MPL、3D-MPL、RC529(例如，參見EP1385541，其在此全文引入作為參考)、PET-脂質A、GLA(糖基吡喃糖脂佐劑，一種合成二糖糖脂；例如，參見US20100310602和US8722064，這兩個專利在此全文引入作為參考)，SLA(例如，參見Carter D等人，Clin. Transl. Immunology 5: e108 (doi:10.1038/cti.2016.63) (2016)，其在此全文引入作為參考，並且其描述了優化用於人類疫苗的TLR4配體的結構-功能方法)，PHAD(磷酸化的六醯基二糖；其結構與GLA相同)、3D-PHAD、3D-(6-醯基)-PHAD (3D(6A)-PHAD)、E6020(CAS Number 287180-63-6)、ONO4007、OM-174等。在任意的實施方案中，所述疫苗組合物的佐劑是包含選自3D-MPL、GLA或SLA的脂質A類似物或衍生物的TLR4激動劑佐劑。在某些實施方案中，脂質類似物或衍生物被配制在脂質體中。MPL and 3D-MPL are lipid A analogs or derivatives that have been modified to reduce lipid A toxicity. Lipids A, MPL, and 3D-MPL have a sugar backbone with long fatty acid chains attached to the sugar backbone, wherein the backbone comprises two 6-carbon sugars in a glycosidic bond and a phosphatyl moiety at position 4. Typically, five to eight long-chain fatty acids (usually 12-14 carbon atoms) are attached to the sugar backbone. Due to derivation from natural sources, MPL or 3D-MPL may exist as a complex or mixture of various fatty acid substitution patterns (eg, heptacyl, hexacyl, pentacyl, etc., with variable fatty acid length). The same is true for some of the other Lipid A analogs or derivatives described herein, however synthetic Lipid A variants can also be defined and homogenized. MPL and its mass production are described in US 4,436,727, which is hereby incorporated by reference in its entirety. 3D-MPL is described in US 4,912,094 B1 (which patent is hereby incorporated by reference in its entirety) and differs from MPL by the selective removal of the 3-hydroxymyristyl residue which is at position 3 with Glucosamine linkage at the reducing end. Examples of lipid A (analogues, derivatives) suitable for use in the vaccine compositions described herein include MPL, 3D-MPL, RC529 (see for example EP1385541, which is hereby incorporated by reference in its entirety), PET-lipid A, GLA (glycosylpyranolipid adjuvant, a synthetic disaccharide glycolipid; see, for example, US20100310602 and US8722064, both of which are hereby incorporated by reference in their entirety), SLA (see, for example, Carter D et al., Clin. Transl. Immunology 5: e108 (doi:10.1038/cti.2016.63) (2016), which is hereby incorporated by reference in its entirety, and which describes a structure-function approach to optimize TLR4 ligands for use in human vaccines), PHAD (phosphorylated Hexacyl disaccharide; its structure is the same as GLA), 3D-PHAD, 3D-(6-acyl)-PHAD (3D(6A)-PHAD), E6020(CAS Number 287180-63-6), ONO4007, OM -174 etc. In any embodiment, the adjuvant of the vaccine composition is a TLR4 agonist adjuvant comprising a lipid A analog or derivative selected from 3D-MPL, GLA or SLA. In certain embodiments, lipid analogs or derivatives are formulated in liposomes.

所述佐劑，優選包括TLR4激動劑，可以以各種方式配制，例如乳劑，例如油包水(w/o)乳劑或水包油(o/w)乳劑(例如MF59、AS03)、穩定(納米)乳劑(SE)、脂質懸浮液、脂質體、(聚合)納米顆粒、病毒體、明礬吸附的、水性制劑(AF)等，代表了用於佐劑中的免疫調節分子和/或針對免疫原的免疫調節分子的各種遞送系統(例如參見Reed等人，Nature Med, 19: 1597-1608 (2013) and Alving CR et al, Curr Opin Immunol 24: 310-315 (2012)，其在此全文引入作為參考)。The adjuvant, preferably including a TLR4 agonist, can be formulated in various ways, such as emulsions, such as water-in-oil (w/o) emulsions or oil-in-water (o/w) emulsions (such as MF59, AS03), stabilized (nano ) emulsions (SE), lipid suspensions, liposomes, (polymeric) nanoparticles, virosomes, alum-adsorbed, aqueous formulations (AF), etc., represent immunomodulatory molecules used in adjuvants and/or against immunogens Various delivery systems for immunomodulatory molecules (see, for example, Reed et al, Nature Med, 19: 1597-1608 (2013) and Alving CR et al, Curr Opin Immunol 24: 310-315 (2012), which are incorporated herein in their entirety as refer to).

在任意的實施方案中，免疫刺激性TLR4激動劑可以任選地與其他免疫調節成分組合，例如角鯊烯水包油乳劑(SE)(例如，MF59、AS03)、皂苷類(如QuilA、QS7、QS21、Matrix M、Iscoms、Iscomatrix等)、鋁鹽、其他TLR的激活劑(如咪唑並喹啉、鞭毛蛋白、dsRNA類似物、TLR9激動劑，如CpG等)、和類似成分(例如參見Reed G. 等人，Nature Med，19: 1597-1608 (2013)，其全部內容通過引用結合於此)。In any of the embodiments, the immunostimulatory TLR4 agonist can optionally be combined with other immunomodulatory ingredients, such as squalene oil-in-water emulsion (SE) (e.g., MF59, AS03), saponins (e.g., QuilA, QS7 , QS21, Matrix M, Iscoms, Iscomatrix, etc.), aluminum salts, activators of other TLRs (such as imidazoquinolines, flagellin, dsRNA analogs, TLR9 agonists, such as CpG, etc.), and similar components (see for example Reed G. et al., Nature Med, 19: 1597-1608 (2013), the entire contents of which are hereby incorporated by reference).

在任意的實施方案中，本文公開的疫苗組合物的佐劑是TLR4激動劑，例如GLA與SE(即GLA-SE)的組合。在任意的實施方案中，本文公開的疫苗組合物的佐劑是TLR4激動劑，如GLA與皂苷(如GLS-QS21)的組合。在任意的實施方案中，上述佐劑可以配制成脂質體。因此，示例性佐劑還包括GLA-LSQ，其被配制成包含合成的TLR4激動劑(例如MPL )和皂苷(例如QS21)的脂質體。In any embodiment, the adjuvant of the vaccine compositions disclosed herein is a TLR4 agonist, such as a combination of GLA and SE (ie, GLA-SE). In any embodiment, the adjuvant of the vaccine compositions disclosed herein is a TLR4 agonist, such as GLA in combination with a saponin (eg, GLS-QS21). In any of the embodiments, the adjuvants described above can be formulated as liposomes. Thus, exemplary adjuvants also include GLA-LSQ formulated as liposomes containing a synthetic TLR4 agonist (eg, MPL®) and a saponin (eg, QS21).

用於本文所述疫苗組合物的其他示例性佐劑包括脂質A類似物或衍生物，包括例如SLA-SE(合成的MPL [SLA]、角鯊烯油/水乳劑)、SLA-納米明礬(合成的MPL[SLA]、鋁鹽)、GLA-納米明礬(合成的MPL[GLA]、鋁鹽)、SLA-AF(合成的MPL[SLA]、水懸浮液)、GLA-AF(合成的MPL[GLA]、水懸浮液)、、SLA-明礬(合成的MPL[SLA]、鋁鹽)、GLA-明礬(合成的MPL[GLA]、鋁鹽)、AS01(MPL、QS21，脂質體)、AS02(MPL、QS21，油/水乳劑)、AS25(MPL，油/水乳劑)、AS04(MPL，鋁鹽)、和AS15(MPL、QS21、CpG，脂質體)。參見例如WO2008/153541、WO2010/141861、WO2013/119856、WO2019/051149、WO 2013/119856、WO2006/116423、US 4,987,237、US 4,436,727、US 4,877,611、US 4,866,034、US 4,912,094、US 4,987,237、US 5,191,072、US 5,593,969、US 6,759,241、US 9,017,698、US 9,149,521、US 9,149,522、US 9,415,097、US 9,415,101、US 9,504,743；Reed G.等，Nature Med, 19: 1597-1608 (2013), Johnson等人，J Med Chem, 42:4640-4649 (1999)，和Ulrich 和 Myers, 1995, Vaccine Design: The Subunit and Adjuvant Approach; Powell and Newman, Eds.; Plenum: New York, 495-524，其全部內容通過引用結合於此。Other exemplary adjuvants for use in the vaccine compositions described herein include lipid A analogs or derivatives including, for example, SLA-SE (synthetic MPL [SLA], squalene oil/water emulsion), SLA-nanoalum ( Synthetic MPL [SLA], aluminum salt), GLA-Nanoalum (synthetic MPL [GLA], aluminum salt), SLA-AF (synthetic MPL [SLA], aqueous suspension), GLA-AF (synthetic MPL [GLA], aqueous suspension), , SLA-alum (synthetic MPL [SLA], aluminum salt), GLA-alum (synthetic MPL [GLA], aluminum salt), AS01 (MPL, QS21, liposomes), AS02 (MPL, QS21, oil/water emulsion), AS25 (MPL, oil/water emulsion), AS04 (MPL, aluminum salt), and AS15 (MPL, QS21, CpG, liposome). See eg WO2008/153541, WO2010/141861, WO2013/119856, WO2019/051149, WO 2013/119856, WO2006/116423, US 4,987,237, US 4,436,727, US 4,877,611, US 4, 866,034, US 4,912,094, US 4,987,237, US 5,191,072, US 5,593,969 , US 6,759,241, US 9,017,698, US 9,149,521, US 9,149,522, US 9,415,097, US 9,415,101, US 9,504,743; Reed G. et al., Nature Med, 19: 1597-1608 (2013), Johnson et al. People, J Med Chem, 42:4640 -4649 (1999), and Ulrich and Myers, 1995, Vaccine Design: The Subunit and Adjuvant Approach; Powell and Newman, Eds.; Plenum: New York, 495-524, the entire contents of which are hereby incorporated by reference.

在任意的實施方案中，所述疫苗組合物的LukA和/或LukB蛋白或多肽可以與免疫原性載體分子偶聯。合適的免疫原性載體分子包括但不限於牛血清白蛋白、雞蛋卵清蛋白、匙孔血藍蛋白、破傷風類毒素、白喉類毒素、甲狀腺球蛋白、肺炎球菌莢膜多糖、CRM 197和腦膜炎球菌外膜蛋白。In any of the embodiments, the LukA and/or LukB protein or polypeptide of the vaccine composition may be conjugated to an immunogenic carrier molecule. Suitable immunogenic carrier molecules include, but are not limited to, bovine serum albumin, egg ovalbumin, keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid, thyroglobulin, pneumococcal capsular polysaccharide, CRM 197, and meningitis Bacterial outer membrane protein.

疫苗組合物可以進一步包括一種或多種附加的金黃色葡萄球菌抗原，所述抗原選自血清型336多糖抗原、聚集因子A、聚集因子B、纖維蛋白原結合蛋白、膠原結合蛋白、彈性蛋白結合蛋白、MHC類似蛋白、多糖細胞內粘附、β溶血素、Δ溶血素、Panton-Valentine殺白細胞素、殺白細胞素M、脫落毒素A、脫落毒素B、V8蛋白酶、透明質酸裂解酶、脂肪酶、葡激酶、腸毒素、腸毒素超抗原SEA、腸毒素超抗原SAB、中毒性休克綜合征毒素-1、聚-N-琥珀醯β-1→6氨基葡萄糖、過氧化氫酶、β-內醯胺酶、磷壁酸、肽聚糖、青黴素結合蛋白、趨化抑制蛋白、補體抑制劑、Sbi、5型抗原、8型抗原和脂磷壁酸。適用於包含在本文所述疫苗組合物中的其他金黃色葡萄球菌抗原包括但不限於CP5、CP8、Eap、Ebh、Emp、EsaB、EsaC、EsxA、EsxB、EsxAB(融合)、IsdA、IsdB、IsdC、MntC、rTSST-1、rTSST-1v、TSST-1、SasF、vWbp、vWh玻連蛋白結合蛋白、Aaa、Aap、Ant、自溶素氨基葡糖苷酶、自溶素醯胺酶、Can、膠原結合蛋白、Csa1A、EFB、彈性蛋白結合蛋白、EPB、FbpA、纖維蛋白原結合蛋白、纖連蛋白結合蛋白、FhuD、FhuD2、FnbA、FnbB、GehD、HarA、HBP、免疫顯性 ABC 轉運蛋白、IsaA/PisA、層粘連蛋白受體、脂肪酶GehD、MAP、Mg2+轉運蛋白、MHC II類似物、MRPII、NPase、RNA III活化蛋白(RAP)、SasA、SasB、SasC、SasD、SasK、SBI、SEA外毒素、SEB外毒素、mSEB、SitC、Ni ABC轉運蛋白、SitC/MntC/唾液結合蛋白、SsaA、SSP-1、SSP-2、Sta006和Sta011。The vaccine composition may further comprise one or more additional S. aureus antigens selected from the group consisting of serotype 336 polysaccharide antigen, aggregation factor A, aggregation factor B, fibrinogen binding protein, collagen binding protein, elastin binding protein , MHC-like protein, polysaccharide intracellular adhesion, β-hemolysin, Δ-hemolysin, Panton-Valentine leukocidin, leukocidin M, abscistoxin A, abscistoxin B, V8 protease, hyaluronan lyase, lipase , Staphylokinase, enterotoxin, enterotoxin superantigen SEA, enterotoxin superantigen SAB, toxic shock syndrome toxin-1, poly-N-succinyl β-1→6 glucosamine, catalase, β-endo Amidase, teichoic acid, peptidoglycan, penicillin-binding protein, chemoattractant protein, complement inhibitor, Sbi, type 5 antigen, type 8 antigen, and lipoteichoic acid. Other S. aureus antigens suitable for inclusion in the vaccine compositions described herein include, but are not limited to, CP5, CP8, Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, EsxAB (fusion), IsdA, IsdB, IsdC , MntC, rTSST-1, rTSST-1v, TSST-1, SasF, vWbp, vWh vitronectin binding protein, Aaa, Aap, Ant, autolysin glucosaminidase, autolysin amidase, Can, collagen Binding protein, Csa1A, EFB, elastin binding protein, EPB, FbpA, fibrinogen binding protein, fibronectin binding protein, FhuD, FhuD2, FnbA, FnbB, GehD, HarA, HBP, immunodominant ABC transporter, IsaA /PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analog, MRPII, NPase, RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SEA toxin, SEB exotoxin, mSEB, SitC, Ni ABC transporter, SitC/MntC/saliva binding protein, SsaA, SSP-1, SSP-2, Sta006 and Sta011.

在任意的實施方案中，所述疫苗組合物被配制成可注射的，其可以例如通過注射裝置(例如注射器或輸注泵)注射。可以通過肌肉內、腹膜內、玻璃體內或靜脈內進行注射。In any of the embodiments, the vaccine composition is formulated to be injectable, which can be injected, for example, via an injection device such as a syringe or an infusion pump. Injections can be given intramuscularly, intraperitoneally, intravitreally or intravenously.

本發明的疫苗組合物可以配制成腸胃外給藥。該組合物的溶液、懸浮液或乳液可以在水中制備，適當地與表面活性劑如羥丙基纖維素混合。分散體也可以在甘油、液體聚乙二醇及其在油中的混合物中制備。示例性的油是石油、動物、植物或合成來源的油，例如花生油、大豆油或礦物油。一般來說，水、鹽水、葡萄糖水溶液和相關的糖溶液，以及二醇，例如丙二醇或聚乙二醇，是優選的液體載體，特別是對於注射溶液。在通常的儲存和使用條件下，這些制劑含有防腐劑以防止微生物的生長。The vaccine compositions of the invention may be formulated for parenteral administration. Solutions, suspensions or emulsions of this composition can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Exemplary oils are oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

適於注射使用的藥物疫苗組合物包括無菌水溶液或分散體以及用於臨時制備無菌注射溶液或分散體的無菌粉末。在所有情況下，劑型必須是無菌的，並且必須是流動性的，達到易於注射的程度。它在生產和儲存條件下必須是穩定的，並且必須防止微生物如細菌和真菌的汙染作用。載體可以是溶劑或分散介質，其含有例如水、乙醇、多元醇(例如甘油、丙二醇和液體聚乙二醇)、及其合適的混合物和植物油。Pharmaceutical vaccine compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the dosage form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

在任意的實施方案中，本文所述的疫苗組合物是固體制劑，例如凍幹或噴霧幹燥的組合物，其可以原樣使用，或者醫生或患者在使用前向其添加溶劑和/或稀釋劑。固體劑型可以包括片劑，例如壓縮片劑和/或包衣片劑，以及膠囊(例如硬或軟明膠膠囊)。疫苗組合物還可以是例如袋裝粉劑、糖衣丸、粉劑、顆粒劑、錠劑或用於重構的粉末的形式。In any embodiment, the vaccine composition described herein is a solid formulation, such as a freeze-dried or spray-dried composition, which can be used as it is, or a solvent and/or diluent can be added to it by a doctor or patient before use. Solid dosage forms may include tablets, such as compressed and/or coated tablets, and capsules, such as hard or soft gelatin capsules. The vaccine composition may also be in the form of, for example, sachets, dragees, powders, granules, lozenges or powders for reconstitution.

疫苗組合物的劑型可以是速釋的，在這種情況下，它們可以包含水溶性或可分散的載體，或者它們可以是延遲釋放、持續釋放或緩和釋放，在這種情況下，它們可以包含可以調節制劑在胃腸道或皮膚下的溶解速率的水不溶性聚合物。The dosage forms of the vaccine compositions may be immediate release, in which case they may comprise a water-soluble or dispersible carrier, or they may be delayed, sustained or modified release, in which case they may comprise Water-insoluble polymers that can modulate the dissolution rate of a formulation in the gastrointestinal tract or under the skin.

在其他實施方案中，所述疫苗組合物可以鼻內、口腔內或舌下遞送。In other embodiments, the vaccine composition may be delivered intranasally, buccally, or sublingually.

疫苗組合物的水性制劑中的酸鹼度可以在3至10之間。在一個實施方案中，所述疫苗組合物的酸鹼度為約7.0至約9.5。在另一個實施方案中，所述疫苗組合物的酸鹼度為約3.0至約7.0。The pH in the aqueous formulation of the vaccine composition may be between 3 and 10. In one embodiment, the pH of the vaccine composition is from about 7.0 to about 9.5. In another embodiment, the pH of the vaccine composition is from about 3.0 to about 7.0.

金黃色葡萄球菌疫苗組合物的用途Uses of staphylococcus aureus vaccine composition

本發明的另一方面涉及本文所述疫苗組合物在預防或抑制受試者的金黃色葡萄球菌感染中的用途。在一個實施方案中，本發明涉及在受試者中產生針對金黃色葡萄球菌的免疫應答的方法，該方法包括在可有效地在所述受試者中產生針對金黃色葡萄球菌的免疫應答的條件下，將本文所述的疫苗組合物施用給受試者。另一個實施方案涉及在有需要的受試者中治療或預防金黃色葡萄球菌感染的方法，包括施用有效量的本文公開的疫苗組合物。另一個實施方案涉及一種在有需要的受試者中再定殖或防止金黃色葡萄球菌的定殖或再定殖的方法，該方法包括施用有效量的本文公開的疫苗組合物。根據這一方面，本文所述的方法適用於在有需要的受試者中防止金黃色葡萄球菌的短期和持續定殖或再定殖。Another aspect of the invention pertains to the use of the vaccine composition described herein for preventing or inhibiting a S. aureus infection in a subject. In one embodiment, the present invention relates to a method of generating an immune response against S. aureus in a subject, the method comprising a method effective to generate an immune response against S. aureus in said subject. Under the conditions, the vaccine composition described herein is administered to the subject. Another embodiment relates to a method of treating or preventing a S. aureus infection in a subject in need thereof comprising administering an effective amount of a vaccine composition disclosed herein. Another embodiment is directed to a method of recolonizing or preventing the colonization or recolonization of S. aureus in a subject in need thereof comprising administering an effective amount of a vaccine composition disclosed herein. According to this aspect, the methods described herein are suitable for preventing short-term and sustained colonization or recolonization by S. aureus in a subject in need thereof.

這些方法包括將本文所述的任何一種疫苗組合物施用於有此需要的受試者，例如有金黃色葡萄球菌暴露或感染風險的受試者。在一個實施方案中，疫苗組合物包含LukA變異多肽(即SEQ ID NO:1或2的變體)和如上所述的野生型LukB蛋白或多肽。在另一個實施方案中，疫苗組合物包含如上所述的野生型LukA蛋白或多肽和LukB變異多肽(即SEQ ID NO:15或16的變體)。在另一個實施方案中，疫苗組合物包含如上所述的LukA變異多肽和LukB變異多肽。根據本發明的這一方面，適合治療的受試者是有發展成金黃色葡萄球菌感染風險的受試者。These methods include administering any of the vaccine compositions described herein to a subject in need thereof, eg, a subject at risk of S. aureus exposure or infection. In one embodiment, the vaccine composition comprises a LukA variant polypeptide (ie, a variant of SEQ ID NO: 1 or 2) and a wild-type LukB protein or polypeptide as described above. In another embodiment, the vaccine composition comprises a wild-type LukA protein or polypeptide as described above and a LukB variant polypeptide (ie, a variant of SEQ ID NO: 15 or 16). In another embodiment, the vaccine composition comprises a LukA variant polypeptide and a LukB variant polypeptide as described above. According to this aspect of the invention, a subject suitable for treatment is a subject at risk of developing a S. aureus infection.

根據本發明的這一方面，向受試者施用預防有效量的疫苗組合物，以產生針對金黃色葡萄球菌感染的免疫應答。預防有效量是能夠產生或引發體液(即抗體介導的)和細胞(T細胞)免疫應答所必需的量。誘導的體液應答足以預防或至少減少金黃色葡萄球菌感染的程度，否則在沒有這種應答的情況下會惡化。優選地，施用預防有效量的本文所述疫苗組合物在受試者中誘導針對金黃色葡萄球菌的中和免疫應答。為了在受試者中實現有效的免疫應答，組合物可以進一步包含一種或多種額外的金黃色葡萄球菌抗原或如上所述的佐劑。在另一個實施方案中，佐劑與組合物分開給予受試者，在給予本發明的組合物之前、之後或同時給予。According to this aspect of the invention, a prophylactically effective amount of the vaccine composition is administered to a subject to generate an immune response against S. aureus infection. A prophylactically effective amount is that amount necessary to generate or elicit humoral (ie, antibody-mediated) and cellular (T-cell) immune responses. The induced humoral response is sufficient to prevent or at least reduce the extent of S. aureus infection that would otherwise be exacerbated in the absence of such a response. Preferably, administering a prophylactically effective amount of a vaccine composition described herein induces a neutralizing immune response against S. aureus in the subject. To achieve an effective immune response in a subject, the composition may further comprise one or more additional S. aureus antigens or adjuvants as described above. In another embodiment, the adjuvant is administered to the subject separately from the composition, before, after or simultaneously with the composition of the invention.

出於本發明這一方面的目的，目標“受試者”包括任何動物，優選哺乳動物，更優選人。在為了預防、抑制或降低受試者中金黃色葡萄球菌感染和金黃色葡萄球菌定殖的嚴重性而施用疫苗組合物的情況下，目標受試者包括有被金黃色葡萄球菌感染風險的任何受試者。特別易感的受試者包括免疫功能低下的嬰兒、青少年、成人和老年人。然而，任何有金黃色葡萄球菌感染風險的嬰兒、少年、成人或老年成人都可以根據本文所述的方法和疫苗組合物進行治療。特別合適的受試者包括有感染耐甲氧西林金黃色葡萄球菌(MRSA)或甲氧西林敏感金黃色葡萄球菌(MSSA)風險的受試者。其他合適的受試者包括那些可能患有或有風險患有由金黃色葡萄球菌感染引起的疾病，即金黃色葡萄球菌相關疾病的受試者，例如皮膚創傷和感染、組織膿腫、毛囊炎、骨髓炎、肺炎、燙傷樣皮膚綜合征、敗血症、膿毒性關節炎、心肌炎、心內膜炎和中毒性休克綜合征。For the purposes of this aspect of the invention, a "subject" of interest includes any animal, preferably a mammal, more preferably a human. Where the vaccine composition is administered for the purpose of preventing, inhibiting or reducing the severity of Staphylococcus aureus infection and Staphylococcus aureus colonization in a subject, the target subject includes any person at risk of being infected by Staphylococcus aureus subject. Particularly susceptible subjects include immunocompromised infants, adolescents, adults and the elderly. However, any infant, juvenile, adult or geriatric adult at risk for S. aureus infection can be treated according to the methods and vaccine compositions described herein. Particularly suitable subjects include those at risk of infection with methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA). Other suitable subjects include those who may have or are at risk of having a disease caused by S. aureus infection, ie, a S. aureus-associated disease, such as skin trauma and infection, tissue abscesses, folliculitis, bone marrow inflammation, pneumonia, scalded skin syndrome, sepsis, septic arthritis, myocarditis, endocarditis, and toxic shock syndrome.

在任何實施方案中，受試者至少或最多 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40、45、50、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、85 或 90 歲（或其中可推導出的任何範圍）。在某些實施方案中，本文描述的受試者或患者，例如人類受試者，是兒科受試者。兒科受試者被定義為小於18歲。在任意的實施方案中，兒科受試者為2歲或更小。在任意的實施方案中，兒科受試者小於1歲。在任意的實施方案中，兒科受試者小於6個月。在任意的實施方案中，兒科受試者為2個月或更小。在任意的實施方案中，人類患者為65歲或以上。在任意的實施方案中，人類患者是衛生保健工作者。在任意的實施方案中，患者是將接受外科手術的患者。In any embodiment, the subject is at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, or 90 years old (or any range derivable therein). In certain embodiments, a subject or patient described herein, eg, a human subject, is a pediatric subject. Pediatric subjects were defined as less than 18 years of age. In any embodiment, the pediatric subject is 2 years or younger. In any embodiment, the pediatric subject is less than 1 year old. In any embodiment, the pediatric subject is less than 6 months old. In any embodiment, the pediatric subject is 2 months or younger. In any of the embodiments, the human patient is 65 years or older. In any of the embodiments, the human patient is a health care worker. In any of the embodiments, the patient is a patient about to undergo surgery.

在可有效誘導強免疫反應的條件下施用疫苗組合物時，也可以考慮許多其他因素。這些因素包括但不限於，例如，組合物中活性劑的濃度、給藥方式和頻率，以及受試者的具體情況，例如年齡、體重和整體健康和免疫狀況。例如，在國際協調會議的出版物和雷明頓制藥科學(Mack Publishing Company 1990)的出版物中可以找到一般性的指導，在此將其全文引入作為參考。臨床醫生可以施用本文所述的疫苗組合物，直到達到所期望或所需預防效果的劑量，例如所需的抗體滴度。預防性反應的進展可以很容易地通過常規檢測來監測。Many other factors may also be considered when administering a vaccine composition under conditions effective to induce a strong immune response. These factors include, but are not limited to, for example, the concentration of the active agent in the composition, the mode and frequency of administration, and the particular conditions of the subject, such as age, weight and general health and immune status. General guidance can be found, for example, in publications of the International Conference on Harmonization and in publications of Remington Pharmaceutical Sciences (Mack Publishing Company 1990), which are hereby incorporated by reference in their entirety. A clinician can administer the vaccine compositions described herein until a dose is achieved for a desired or desired prophylactic effect, eg, a desired antibody titer. The progress of the preventive response can be easily monitored by routine testing.

在本發明的一個實施方案中，本文所述的疫苗組合物可以以預防性的方式施用，以預防、延遲或抑制有感染金黃色葡萄球菌病毒風險或有發展成相關病症風險的受試者中金黃色葡萄球菌病毒感染的發展。在本發明的任何實施方案中，疫苗組合物的預防性給藥可有效地完全預防個體中的金黃色葡萄球菌感染。在其他實施方案中，預防性給藥可有效預防在沒有這種給藥的情況下會發生感染的全部程度，即基本上預防或抑制個體中的金黃色葡萄球菌感染。In one embodiment of the invention, the vaccine compositions described herein may be administered in a prophylactic manner to prevent, delay or inhibit the development of S. aureus virus infection in subjects at risk of infection or development of related conditions. Development of Staphylococcus aureus virus infection. In any of the embodiments of the invention, prophylactic administration of the vaccine composition is effective to completely prevent S. aureus infection in an individual. In other embodiments, prophylactic administration is effective to prevent the full extent of infection that would have occurred in the absence of such administration, ie, substantially prevent or inhibit a S. aureus infection in an individual.

在使用預防性組合物預防金黃色葡萄球菌感染的情況下，該組合物的劑量應足以產生能夠中和金黃色葡萄球菌LukAB介導的細胞毒性的抗體滴度，並能夠減少許多症狀，降低至少一種症狀的嚴重性，或延緩至少一種症狀的進一步發展，甚至完全緩解感染。In cases where a prophylactic composition is used to prevent S. aureus infection, the dose of the composition should be sufficient to generate antibody titers capable of neutralizing S. aureus LukAB-mediated cytotoxicity and capable of reducing a number of symptoms, reducing at least Severity of one symptom, or delay of further development of at least one symptom, or even complete remission of the infection.

本文所述疫苗組合物的預防有效量將取決於是否共同施用佐劑，在沒有佐劑的情況下需要更高的劑量。每名患者施用的變異LukA和/或LukB的量可以在1 μg-500 μg之間。在任何實施方案中，每次人體注射使用5、10、20、25、50或100 μg。偶爾，每次注射使用1-50mg的較高劑量。通常，每次人體注射使用約10、20、30、40或50mg。注射的時間可以有很大的不同，從一年一次到十年一次。通常，有效劑量可以通過從受試者獲得流體樣品(通常是血清樣品)，並確定針對LukA、LukB或LukB產生的抗體的滴度來監測，所述滴度通過使用本領域公知的並且易於適用於待測特定抗原的方法進行測定。理想情況下，在首次給藥前采集樣本，並在每次免疫接種後采集後續樣本並進行滴度測定。通常，在血清稀釋度為1:100時，提供比對照或“背景”水平至少大四倍的可檢測滴度的劑量或給藥方案是理想的，其中背景是相對於對照血清或相對於酶聯免疫吸附試驗中的平板背景來定義的。A prophylactically effective amount of the vaccine composition described herein will depend on whether or not an adjuvant is co-administered, in which case higher doses will be required. The amount of variant LukA and/or LukB administered per patient may be between 1 μg-500 μg. In any embodiment, 5, 10, 20, 25, 50 or 100 μg is used per human injection. Occasionally, higher doses of 1-50 mg per injection are used. Typically, about 10, 20, 30, 40 or 50 mg will be used per human injection. The timing of injections can vary widely, from once a year to once a decade. Typically, the effective dose can be monitored by obtaining a fluid sample (usually a serum sample) from the subject and determining the titer of antibodies raised against LukA, LukB, or LukB using methods known in the art and readily applicable Determination is carried out in the method for the specific antigen to be tested. Ideally, samples are collected before the first dose and subsequent samples are collected and titered after each immunization. Typically, a dose or dosing regimen that provides a detectable titer at least four-fold greater than control or "background" levels at a serum dilution of 1:100, where background is relative to control serum or relative to enzyme plate background in linked immunosorbent assays.

本發明的疫苗組合物可以通過胃腸外、局部、靜脈內、口服、腹膜內、鼻內或肌內方式給藥，用於預防性治療。The vaccine composition of the present invention may be administered parenterally, topically, intravenously, orally, intraperitoneally, intranasally or intramuscularly for prophylactic treatment.

適於注射使用的藥物疫苗制劑包括無菌水溶液或分散體以及用於臨時制備無菌注射溶液或分散體的無菌粉末。在所有情況下，劑型必須是無菌的，並且必須是流動性的，達到易於注射的程度。它在生產和儲存條件下必須是穩定的，並且必須能夠防止微生物如細菌和真菌的汙染。載體可以是溶劑或分散介質，其含有例如水、乙醇、多元醇(例如甘油、丙二醇和液體聚乙二醇)、其合適的混合物和植物油。Pharmaceutical vaccine formulations suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the dosage form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

實施方案implementation plan

本發明提供了以下非限制性實施方案。The invention provides the following non-limiting embodiments.

實施方案1是SEQ ID NO:25變異的金黃色葡萄球菌殺白細胞素A (LukA)多肽，所述LukA變異多肽包含:對應於SEQ ID NO:25的氨基酸殘基Lys83、Ser141、Val113和Val193的一個或多個氨基酸殘基上的氨基酸取代。Embodiment 1 is a variant Staphylococcus aureus leukocidin A (LukA) polypeptide of SEQ ID NO:25, said LukA variant polypeptide comprising: corresponding to amino acid residues Lys83, Ser141, Val113 and Val193 of SEQ ID NO:25 Amino acid substitutions at one or more amino acid residues.

實施方案2是實施方案1所述的LukA變異多肽，其中所述LukA變異多肽在對應於SEQ ID NO:25的Glu323的氨基酸殘基上進一步包含氨基酸取代。Embodiment 2 is the LukA variant polypeptide of embodiment 1, wherein the LukA variant polypeptide further comprises an amino acid substitution at the amino acid residue corresponding to Glu323 of SEQ ID NO:25.

實施方案3是實施方案2所述的LukA變異多肽，其中對應於Glu323的氨基酸殘基上的氨基酸取代包括穀氨酸到丙氨酸(Glu323Ala)的取代。Embodiment 3 is the LukA variant polypeptide of embodiment 2, wherein the amino acid substitution at the amino acid residue corresponding to Glu323 comprises a glutamic acid to alanine (Glu323Ala) substitution.

實施方案4是實施方案1-3中任一項所述的LukA變異多肽，其中對應於Lys83的氨基酸殘基上的氨基酸取代包括賴氨酸到甲硫氨酸(Lys83Met)的取代。Embodiment 4 is the LukA variant polypeptide of any one of embodiments 1-3, wherein the amino acid substitution at the amino acid residue corresponding to Lys83 comprises a lysine to methionine (Lys83Met) substitution.

實施方案5是實施方案1-4中任一項所述的LukA變異多肽，其中對應於Ser141的氨基酸殘基上的氨基酸取代包括絲氨酸到丙氨酸(Ser141Ala)的取代。Embodiment 5 is the LukA variant polypeptide of any one of embodiments 1-4, wherein the amino acid substitution at the amino acid residue corresponding to Ser141 comprises a serine to alanine (Ser141Ala) substitution.

實施方案6是實施方案1-5中任一項所述的LukA變異多肽，其中對應於Val113的氨基酸殘基上的氨基酸取代包括纈氨酸到異亮氨酸(Val113Ile)的取代。Embodiment 6 is the LukA variant polypeptide of any one of embodiments 1-5, wherein the amino acid substitution at the amino acid residue corresponding to Val113 comprises a substitution of valine to isoleucine (Val113Ile).

實施方案7是實施方案1-6中任一項所述的LukA變異多肽，其中對應於Val193的氨基酸殘基上的氨基酸取代包括纈氨酸到異亮氨酸(Val193Ile)的取代。Embodiment 7 is the LukA variant polypeptide of any one of embodiments 1-6, wherein the amino acid substitution at the amino acid residue corresponding to Val193 comprises a substitution of valine to isoleucine (Val193Ile).

實施方案8是實施方案2-7中任一項所述的LukA變異多肽，其中所述LukA變異多肽包含對應於SEQ ID NO:25的氨基酸殘基Lys83、Ser141、Val113、Val193和Glu323的每個氨基酸殘基上的氨基酸取代。Embodiment 8 is the LukA variant polypeptide of any one of embodiments 2-7, wherein the LukA variant polypeptide comprises each of amino acid residues Lys83, Ser141, Val113, Val193, and Glu323 corresponding to SEQ ID NO:25 Amino acid substitutions on amino acid residues.

實施方案9是實施方案8所述的LukA變異多肽，其中氨基酸取代包括Lys83Met、Ser141Ala、Val113Ile、Val193Ile和Glu323Ala。Embodiment 9 is the LukA variant polypeptide of embodiment 8, wherein the amino acid substitutions include Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala.

實施方案10是實施方案1所述的LukA變異多肽，其中所述變體是SEQ ID NO:1的CC8 LukA變體，包含對應於SEQ ID NO:1中Lys80Met、Ser138Ala、Val110Ile、Val190Ile和Glu320Ala的氨基酸取代。Embodiment 10 is the LukA variant polypeptide of embodiment 1, wherein the variant is a CC8 LukA variant of SEQ ID NO: 1 comprising a protein corresponding to Lys80Met, Ser138Ala, Val110Ile, Val190Ile, and Glu320Ala in SEQ ID NO: 1 Amino acid substitutions.

實施方案11是實施方案10所述的LukA變異多肽，其中所述LukA變異多肽包含SEQ ID NO:3的氨基酸序列。Embodiment 11 is the LukA variant polypeptide of embodiment 10, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3.

實施方案12是實施方案1所述的LukA變異多肽，其中所述變體是SEQ ID NO:2的CC45 LukA變體，包含對應於SEQ ID NO:2中Lys81Met、Ser139Ala、Val111Ile、Val191Ile和Glu321Ala的氨基酸取代。Embodiment 12 is the LukA variant polypeptide of embodiment 1, wherein the variant is the CC45 LukA variant of SEQ ID NO:2 comprising a protein corresponding to Lys81Met, Ser139Ala, Val111Ile, Val191Ile, and Glu321Ala in SEQ ID NO:2 Amino acid substitutions.

實施方案13是實施方案12所述的LukA變異多肽，其中所述LukA變異多肽包含SEQ ID NO:4的氨基酸序列。Embodiment 13 is the LukA variant polypeptide of embodiment 12, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:4.

實施方案14是實施方案1-13中任一項所述的LukA變異多肽，其中所述LukA變異多肽進一步包含對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的一個或多個氨基酸殘基上的氨基酸取代。Embodiment 14 is the LukA variant polypeptide of any one of embodiments 1-13, wherein the LukA variant polypeptide further comprises one or more of amino acid residues Tyr74, Asp140, Gly149, and Gly156 corresponding to SEQ ID NO:25 Amino acid substitutions on amino acid residues.

實施方案15是實施方案14所述的LukA變異多肽，其中對應於Tyr74的氨基酸殘基處的氨基酸取代包括酪氨酸至半胱氨酸(Tyr74Cys)的取代。Embodiment 15 is the LukA variant polypeptide of embodiment 14, wherein the amino acid substitution at the amino acid residue corresponding to Tyr74 comprises a tyrosine to cysteine (Tyr74Cys) substitution.

實施方案16是實施方案14或15所述的LukA變異多肽，其中對應於Asp140的氨基酸殘基上的氨基酸取代包括天冬醯胺到半胱氨酸(Asp140Cys)的取代。Embodiment 16 is the LukA variant polypeptide of embodiment 14 or 15, wherein the amino acid substitution at the amino acid residue corresponding to Aspl40 comprises an asparagine to cysteine (Asp140Cys) substitution.

實施方案17是實施方案14-16中任一項所述的LukA變異多肽，其中對應於Gly149的氨基酸殘基上的氨基酸取代包括甘氨酸到半胱氨酸(Gly149Cys)的取代。Embodiment 17 is the LukA variant polypeptide of any one of embodiments 14-16, wherein the amino acid substitution at the amino acid residue corresponding to Gly149 comprises a glycine to cysteine (Gly149Cys) substitution.

實施方案18是實施方案14-17中任一項所述的LukA變異多肽，其中對應於Gly156的氨基酸殘基上的氨基酸取代包括甘氨酸到半胱氨酸(Gly156Cys)的取代。Embodiment 18 is the LukA variant polypeptide of any one of embodiments 14-17, wherein the amino acid substitution at the amino acid residue corresponding to Gly156 comprises a glycine to cysteine (Gly156Cys) substitution.

實施方案19是實施方案14-18中任一項所述的LukA變異多肽，其中所述LukA變異多肽包含對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的每個氨基酸殘基上的氨基酸取代。Embodiment 19 is the LukA variant polypeptide of any one of embodiments 14-18, wherein the LukA variant polypeptide comprises each amino acid residue corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO:25 Amino acid substitutions on the base.

實施方案20是實施方案14所述的LukA變異多肽，其中所述變體是SEQ ID NO:1的CC8 LukA變體，包含對應於SEQ ID NO:1的Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Glu320Ala、Tyr71Cys、Asp137Cys、Gly146Cys和Gly153Cys的氨基酸取代。Embodiment 20 is the LukA variant polypeptide of embodiment 14, wherein the variant is the CC8 LukA variant of SEQ ID NO:1 comprising Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Glu320Ala, Amino acid substitutions of Tyr71Cys, Asp137Cys, Gly146Cys and Gly153Cys.

實施方案21是實施方案20所述的LukA變異多肽，其中所述LukA變異多肽包含SEQ ID NO:5的氨基酸序列。Embodiment 21 is the LukA variant polypeptide of embodiment 20, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:5.

實施方案22是實施方案14所述的LukA變異多肽，其中所述變體是SEQ ID NO:2的CC45 LukA變體，包含對應於SEQ ID NO:2的Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Glu321Ala、Tyr72Cys、Asp138Cys、Gly147Cys和Gly154Cys的氨基酸取代。Embodiment 22 is the LukA variant polypeptide of embodiment 14, wherein the variant is the CC45 LukA variant of SEQ ID NO:2 comprising Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Glu321Ala, Amino acid substitutions of Tyr72Cys, Asp138Cys, Gly147Cys and Gly154Cys.

實施方案23是實施方案22所述的LukA變異多肽，其中所述LukA變異多肽包含SEQ ID NO:6的氨基酸序列。Embodiment 23 is the LukA variant polypeptide of embodiment 22, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:6.

實施方案24是實施方案1-23中任一項所述的LukA變異多肽，其中所述LukA變異多肽在對應於SEQ ID NO:25的氨基酸殘基Thr249的氨基酸殘基上進一步包含氨基酸取代。Embodiment 24 is the LukA variant polypeptide of any one of embodiments 1-23, wherein the LukA variant polypeptide further comprises an amino acid substitution at an amino acid residue corresponding to amino acid residue Thr249 of SEQ ID NO:25.

實施方案25是實施方案24所述的LukA變異多肽，其中對應於Thr249的氨基酸殘基上的氨基酸取代包括蘇氨酸到纈氨酸(Thr249Val)的取代。Embodiment 25 is the LukA variant polypeptide of embodiment 24, wherein the amino acid substitution at the amino acid residue corresponding to Thr249 comprises a threonine to valine (Thr249Val) substitution.

實施方案26是實施方案25所述的LukA變異多肽，其中所述變異LukA蛋白包含SEQ ID NO:7或SEQ ID NO:8的氨基酸序列。Embodiment 26 is the variant LukA polypeptide of embodiment 25, wherein the variant LukA protein comprises the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

實施方案27是實施方案1-26中任一項所述的LukA變異多肽，進一步包含氨基末端信號序列。Embodiment 27 is the LukA variant polypeptide of any one of embodiments 1-26, further comprising an amino terminal signal sequence.

實施方案28是實施方案27所述的LukA變異多肽，其中氨基末端信號序列包含SEQ ID NO:23的氨基酸序列。Embodiment 28 is the LukA variant polypeptide of embodiment 27, wherein the amino-terminal signal sequence comprises the amino acid sequence of SEQ ID NO:23.

實施方案29是實施方案1-28中任一項所述的LukA變異多肽，其進一步包含氨基末端純化序列。Embodiment 29 is the LukA variant polypeptide of any one of embodiments 1-28, further comprising an amino-terminal purification sequence.

實施方案30是一種編碼實施方案1-29中任一項所述的LukA變異多肽的核酸分子。Embodiment 30 is a nucleic acid molecule encoding the LukA variant polypeptide of any one of Embodiments 1-29.

實施方案31是一種包含實施方案30所述的核酸分子的表達載體。Embodiment 31 is an expression vector comprising the nucleic acid molecule of embodiment 30.

實施方案32是一種包含實施方案31所述的表達載體的宿主細胞。Embodiment 32 is a host cell comprising the expression vector of embodiment 31.

實施方案33是SEQ ID NO:39變異的金黃色葡萄球菌殺白細胞素B (LukB)蛋白或多肽，所述LukB變異多肽包含對應於SEQ ID NO:39的氨基酸殘基Val53的氨基酸殘基上的氨基酸取代。Embodiment 33 is a variant Staphylococcus aureus leukocidin B (LukB) protein or polypeptide of SEQ ID NO:39, the LukB variant polypeptide comprising an amino acid residue corresponding to amino acid residue Val53 of SEQ ID NO:39 Amino acid substitutions.

實施方案34是實施方案33所述的LukB變異多肽，其中在對應於Val53的氨基酸殘基上的氨基酸取代包括纈氨酸到亮氨酸(Val53Leu)的取代。Embodiment 34 is the LukB variant polypeptide of embodiment 33, wherein the amino acid substitution at the amino acid residue corresponding to Val53 comprises a valine to leucine (Val53Leu) substitution.

實施方案35是實施方案33所述的LukB變異多肽，其中所述變異體是SEQ ID NO:15的CC8 LukB變體，包含對應於SEQ ID NO:15的Val53Leu的氨基酸取代。Embodiment 35 is the LukB variant polypeptide of embodiment 33, wherein the variant is the CC8 LukB variant of SEQ ID NO:15 comprising an amino acid substitution corresponding to Val53Leu of SEQ ID NO:15.

實施方案36是實施方案35所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:17的氨基酸序列。Embodiment 36 is the LukB variant polypeptide of embodiment 35, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:17.

實施方案37是實施方案33所述的LukB變異多肽，其中所述變體是SEQ ID NO:16的CC45 LukB變異體，包含對應於SEQ ID NO:16的Val53Leu的氨基酸取代。Embodiment 37 is the LukB variant polypeptide of embodiment 33, wherein the variant is the CC45 LukB variant of SEQ ID NO:16 comprising an amino acid substitution corresponding to Val53Leu of SEQ ID NO:16.

實施方案38是實施方案37所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:18的氨基酸序列。Embodiment 38 is the LukB variant polypeptide of embodiment 37, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:18.

實施方案39是實施方案33或34所述的LukB變異多肽，其中所述變異體進一步包含對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的一個或多個氨基酸殘基上的氨基酸取代。Embodiment 39 is the LukB variant polypeptide of embodiment 33 or 34, wherein the variant further comprises one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39 amino acid substitutions.

實施方案40是實施方案39所述的LukB變異多肽，其中對應於Glu45的氨基酸殘基上的氨基酸取代包括穀氨酸到半胱氨酸(Glu45Cys)的取代。Embodiment 40 is the LukB variant polypeptide of embodiment 39, wherein the amino acid substitution at the amino acid residue corresponding to Glu45 comprises a glutamic acid to cysteine (Glu45Cys) substitution.

實施方案41是實施方案39或40所述的LukB變異多肽，其中對應於Glu109的氨基酸殘基上的氨基酸取代包括穀氨酸到半胱氨酸(Glu109Cys)的取代。Embodiment 41 is the LukB variant polypeptide of embodiment 39 or 40, wherein the amino acid substitution at the amino acid residue corresponding to Glu109 comprises a glutamic acid to cysteine (Glu109Cys) substitution.

實施方案42是實施方案39-41中任一項所述的LukB變異多肽，其中對應於Thr121的氨基酸殘基上的氨基酸取代包括蘇氨酸到半胱氨酸(Thr121Cys)的取代。Embodiment 42 is the LukB variant polypeptide of any one of embodiments 39-41, wherein the amino acid substitution at the amino acid residue corresponding to Thr121 comprises a threonine to cysteine (Thr121Cys) substitution.

實施方案43是實施方案39-42中任一項所述的LukB變異多肽，其中對應於Arg154的氨基酸殘基上的氨基酸取代包括精氨酸到半胱氨酸(Arg154Cys)的取代。Embodiment 43 is the LukB variant polypeptide of any one of embodiments 39-42, wherein the amino acid substitution at the amino acid residue corresponding to Arg154 comprises an arginine to cysteine (Arg154Cys) substitution.

實施方案44是實施方案39-43中任一項所述的LukB變異多肽，其中所述LukB變異多肽包含對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上的氨基酸取代。Embodiment 44 is the LukB variant polypeptide of any one of embodiments 39-43, wherein the LukB variant polypeptide comprises each of amino acid residues Glu45, Glu109, Thr121, and Arg154 corresponding to SEQ ID NO:39 Amino acid substitutions on the base.

實施方案45是實施方案39所述的LukB變異多肽，其中所述變體是SEQ ID NO:15的CC8 LukB變體，包含對應於SEQ ID NO:15的Val53Leu、Glu45Cys、Glu109Cys、Thr121Cys和Arg154Cys的氨基酸取代。Embodiment 45 is the LukB variant polypeptide of embodiment 39, wherein the variant is the CC8 LukB variant of SEQ ID NO: 15 comprising Val53Leu, Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys corresponding to SEQ ID NO: 15 Amino acid substitutions.

實施方案46是實施方案45所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:19的氨基酸序列。Embodiment 46 is the LukB variant polypeptide of embodiment 45, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:19.

實施方案47是實施方案39所述的LukB變異多肽，其中所述變體是SEQ ID NO:16的CC45 LukB變體，包含對應於SEQ ID NO:16的Val53Leu、Glu45Cys、Glu110Cys、Thr123Cys和Arg155Cys的氨基酸取代。Embodiment 47 is the LukB variant polypeptide of embodiment 39, wherein the variant is the CC45 LukB variant of SEQ ID NO: 16 comprising Val53Leu, Glu45Cys, Glu110Cys, Thr123Cys, and Arg155Cys corresponding to SEQ ID NO: 16 Amino acid substitutions.

實施方案48是實施方案47所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:20的氨基酸序列。Embodiment 48 is the LukB variant polypeptide of embodiment 47, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:20.

實施方案49是SEQ ID NO:39變異的金黃色葡萄球菌殺白細胞素B (LukB)蛋白或多肽，所述LukB變異多肽包含:對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的一個或多個氨基酸殘基上的氨基酸取代。Embodiment 49 is a variant Staphylococcus aureus leukocidin B (LukB) protein or polypeptide of SEQ ID NO:39, said LukB variant polypeptide comprising: corresponding to amino acid residues Glu45, Glu109, Thr121 and Amino acid substitutions at one or more amino acid residues of Arg154.

實施方案50是實施方案49所述的LukB變異多肽，其中對應於Glu45的氨基酸殘基上的氨基酸取代包括穀氨酸到半胱氨酸(Glu45Cys)的取代，對應於Thr121的氨基酸殘基上的氨基酸取代包括蘇氨酸到半胱氨酸(Thr121Cys)的取代。Embodiment 50 is the LukB variant polypeptide of embodiment 49, wherein the amino acid substitution at the amino acid residue corresponding to Glu45 comprises a glutamic acid to cysteine (Glu45Cys) substitution, the amino acid residue corresponding to Thr121 is Amino acid substitutions included threonine to cysteine (Thrl21Cys).

實施方案51是實施方案49或50所述的LukB變異多肽，其中對應於Glu109的氨基酸殘基上的氨基酸取代包括穀氨酸到半胱氨酸(Glu109Cys)的取代，對應於Arg154的氨基酸殘基上的氨基酸取代包括精氨酸到半胱氨酸(Arg154Cys)的取代。Embodiment 51 is the LukB variant polypeptide of embodiment 49 or 50, wherein the amino acid substitution at the amino acid residue corresponding to Glu109 comprises a glutamic acid to cysteine (Glu109Cys) substitution, the amino acid residue corresponding to Arg154 Amino acid substitutions on include an arginine to cysteine (Arg154Cys) substitution.

實施方案52是實施方案49-51中任一項所述的LukB變異多肽，其中所述LukB變異多肽包含在對應於SEQ ID NO:39的氨基酸Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上的氨基酸取代。Embodiment 52 is the LukB variant polypeptide of any one of embodiments 49-51, wherein the LukB variant polypeptide is comprised at each of the amino acid residues corresponding to amino acids Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO:39 amino acid substitutions.

實施方案53是實施方案52所述的LukB變異多肽，其中所述變體是SEQ ID NO:15的CC8 LukB變體，包含對應於SEQ ID NO:15的Glu45Cys、Glu109Cys、Thr121Cys和Arg154Cys的氨基酸取代。Embodiment 53 is the LukB variant polypeptide of embodiment 52, wherein the variant is the CC8 LukB variant of SEQ ID NO: 15 comprising amino acid substitutions corresponding to Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys of SEQ ID NO: 15 .

實施方案54是實施方案53所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:21的氨基酸序列。Embodiment 54 is the LukB variant polypeptide of embodiment 53, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:21.

實施方案55是實施方案52所述的LukB變異多肽，其中所述變體是SEQ ID NO:16的CC45 LukB變體，包含對應於SEQ ID NO:16的Glu45Cys、Glu110Cys、Thr123Cys和Arg155Cys的氨基酸取代。Embodiment 55 is the LukB variant polypeptide of embodiment 52, wherein the variant is the CC45 LukB variant of SEQ ID NO: 16 comprising amino acid substitutions corresponding to Glu45Cys, Glu110Cys, Thr123Cys, and Arg155Cys of SEQ ID NO: 16 .

實施方案56是實施方案55所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:22的氨基酸序列。Embodiment 56 is the LukB variant polypeptide of embodiment 55, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:22.

實施方案57是實施方案33-56中任一項所述的LukB變異多肽，進一步包含:氨基末端信號序列。Embodiment 57 is the LukB variant polypeptide of any one of embodiments 33-56, further comprising: an amino terminal signal sequence.

實施方案58是實施方案57所述的LukB變異多肽，其中氨基末端信號序列包含SEQ ID NO:23的氨基酸序列。Embodiment 58 is the LukB variant polypeptide of embodiment 57, wherein the amino-terminal signal sequence comprises the amino acid sequence of SEQ ID NO:23.

實施方案59是實施方案33-58中任一項所述的LukB變異多肽，進一步包含:氨基末端純化標簽。Embodiment 59 is the LukB variant polypeptide of any one of embodiments 33-58, further comprising: an amino-terminal purification tag.

實施方案60是一種編碼實施方案33-59中任一項所述的LukB變異多肽的核酸分子。Embodiment 60 is a nucleic acid molecule encoding the LukB variant polypeptide of any one of embodiments 33-59.

實施方案61是一種包含實施方案60的核酸分子的表達載體。Embodiment 61 is an expression vector comprising the nucleic acid molecule of embodiment 60.

實施方案62是一種包含實施方案30的核酸分子的表達載體，該核酸分子可操作地與實施方案60的核酸分子偶聯。Embodiment 62 is an expression vector comprising the nucleic acid molecule of embodiment 30 operably coupled to the nucleic acid molecule of embodiment 60.

實施方案63是實施方案62所述的表達載體，其包含與SEQ ID NO:55 (CC45 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與和SEQ ID NO:59 (CC45 LukB)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。Embodiment 63 is the expression vector of embodiment 62, comprising at least 85%, at least 90%, at least 95%, at least 97%, or at least A nucleotide sequence with 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 59 (CC45 LukB) having at least 85%, at least 90%, at least 95%, at least 97%, or Nucleotide sequences of at least 99% sequence similarity are operably coupled.

實施方案64是實施方案62所述的表達載體，其包含與SEQ ID NO:55 (CC45 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與和SEQ ID NO:61 (CC45 LukB變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。Embodiment 64 is the expression vector of embodiment 62, comprising at least 85%, at least 90%, at least 95%, at least 97%, or at least A 99% sequence similarity to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97% to the nucleotide sequence of SEQ ID NO: 61 (CC45 LukB variant) % or at least 99% sequence similarity of nucleotide sequences are operably coupled.

實施方案65是實施方案62所述的表達載體，其包含與SEQ ID NO:54 (CC8 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與和SEQ ID NO:58 (CC8 LukB)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。Embodiment 65 is the expression vector of embodiment 62, comprising at least 85%, at least 90%, at least 95%, at least 97%, or at least A nucleotide sequence with 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 58 (CC8 LukB) having at least 85%, at least 90%, at least 95%, at least 97%, or Nucleotide sequences of at least 99% sequence similarity are operably coupled.

實施方案66是實施方案62所述的表達載體，其包含與SEQ ID NO:54 (CC8 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與和SEQ ID NO:61 (CC45 LukB變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。Embodiment 66 is the expression vector of embodiment 62, comprising at least 85%, at least 90%, at least 95%, at least 97%, or at least A 99% sequence similarity to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97% to the nucleotide sequence of SEQ ID NO: 61 (CC45 LukB variant) % or at least 99% sequence similarity of nucleotide sequences are operably coupled.

實施方案67是實施方案62所述的表達載體，其包含與SEQ ID NO:54 (CC8 LukA變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列，所述核苷酸序列與和SEQ ID NO:60 (CC8 LukB變體)的核苷酸序列具有至少85%、至少90%、至少95%、至少97%或至少99%序列相似性的核苷酸序列可操作地偶聯。Embodiment 67 is the expression vector of embodiment 62, comprising at least 85%, at least 90%, at least 95%, at least 97%, or at least A 99% sequence similarity to a nucleotide sequence of at least 85%, at least 90%, at least 95%, at least 97% to the nucleotide sequence of SEQ ID NO: 60 (CC8 LukB variant) % or at least 99% sequence similarity of nucleotide sequences are operably coupled.

實施方案68是包含實施方案61-67中任一項所述的表達載體的宿主細胞。Embodiment 68 is a host cell comprising the expression vector of any one of embodiments 61-67.

實施方案69是包含實施方案61-67中任一項所述的表達載體的金黃色葡萄球菌疫苗組合物。Embodiment 69 is a S. aureus vaccine composition comprising the expression vector of any one of embodiments 61-67.

實施方案70是一種金黃色葡萄球菌疫苗組合物，其包含實施方案1-29中任一項所述的一種或多種LukA變異多肽。Embodiment 70 is a S. aureus vaccine composition comprising one or more LukA variant polypeptides of any one of embodiments 1-29.

實施方案71是實施方案70所述的疫苗組合物，其中LukA變異多肽是SEQ ID NO:1的變體。Embodiment 71 is the vaccine composition of embodiment 70, wherein the LukA variant polypeptide is a variant of SEQ ID NO:1.

實施方案72是實施方案70或71所述的疫苗組合物，其進一步包含:殺白細胞素B (LukB)蛋白或多肽，所述LukB蛋白或多肽與SEQ ID NO:15的氨基酸序列具有至少85%的序列相似性。Embodiment 72 is the vaccine composition of embodiment 70 or 71, further comprising: a leukocidin B (LukB) protein or polypeptide having at least 85% of the amino acid sequence of SEQ ID NO: 15 sequence similarity.

實施方案73是實施方案70或71所述的疫苗組合物，其進一步包含:殺白細胞素B (LukB)蛋白或多肽，所述LukB蛋白或多肽與SEQ ID NO:16的氨基酸序列具有至少85%的序列相似性。Embodiment 73 is the vaccine composition of embodiment 70 or 71, further comprising: a leukocidin B (LukB) protein or polypeptide having at least 85% of the amino acid sequence of SEQ ID NO: 16 sequence similarity.

實施方案74是實施方案73所述的疫苗組合物，其中LukA變異多肽在對應於SEQ ID NO:1的氨基酸殘基Lys80、Ser138、Val110、Val190和Glu320Ala的一個或多個氨基酸殘基上包含氨基酸取代。Embodiment 74 is the vaccine composition of embodiment 73, wherein the LukA variant polypeptide comprises amino acids at one or more amino acid residues corresponding to amino acid residues Lys80, Ser138, Val110, Val190, and Glu320Ala of SEQ ID NO:1 replace.

實施方案75是實施方案74所述的疫苗組合物，其中LukA變異多肽包含與SEQ ID NO:3的氨基酸序列具有至少90%序列同一性的氨基酸序列，並且LukB多肽包含與SEQ ID NO:18的氨基酸序列具有至少90%序列同一性的氨基酸序列。Embodiment 75 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3, and the LukB polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:18 Amino acid sequence An amino acid sequence having at least 90% sequence identity.

實施方案76是實施方案74所述的疫苗組合物，其中LukA變異多肽包含SEQ ID NO:3的氨基酸序列，LukB多肽包含SEQ ID NO:18的氨基酸序列。Embodiment 76 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, and the LukB polypeptide comprises the amino acid sequence of SEQ ID NO:18.

實施方案77是實施方案74所述的疫苗組合物，其中LukA變異多肽包含SEQ ID NO:3的氨基酸序列，LukB蛋白或多肽包含SEQ ID NO:22的氨基酸序列。Embodiment 77 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:22.

實施方案78是實施方案74所述的疫苗組合物，其中LukA變異多肽包含SEQ ID NO:3的氨基酸序列，LukB蛋白或多肽包含SEQ ID NO:20的氨基酸序列。Embodiment 78 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:20.

實施方案79是實施方案70所述的疫苗組合物，其中LukA變異多肽是SEQ ID NO:2的變體。Embodiment 79 is the vaccine composition of embodiment 70, wherein the LukA variant polypeptide is a variant of SEQ ID NO:2.

實施方案80是實施方案79所述的疫苗組合物，其進一步包含殺白細胞素B (LukB)蛋白或多肽，所述LukB蛋白或多肽與SEQ ID NO:16的氨基酸序列具有至少85%的序列相似性。Embodiment 80 is the vaccine composition of embodiment 79, further comprising a leukocidin B (LukB) protein or polypeptide having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 16 sex.

實施方案81是實施方案79所述的疫苗組合物，其進一步包含殺白細胞素B (LukB)蛋白或多肽，所述LukB蛋白或多肽與SEQ ID NO:15的氨基酸序列具有至少85%的序列相似性。Embodiment 81 is the vaccine composition of embodiment 79, further comprising a leukocidin B (LukB) protein or polypeptide having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 15 sex.

實施方案82是實施方案79-81中任一項所述的疫苗組合物，其中所述LukA變異多肽包含對應於SEQ ID NO:2的氨基酸殘基Lys81、Ser139、Val111、Val191和Glu321Ala的一個或多個氨基酸殘基上的氨基酸取代。Embodiment 82 is the vaccine composition of any one of embodiments 79-81, wherein the LukA variant polypeptide comprises one or more of amino acid residues Lys81, Ser139, Val111, Val191, and Glu321Ala corresponding to SEQ ID NO:2 Amino acid substitutions at multiple amino acid residues.

實施方案83是實施方案82所述的疫苗組合物，其中LukA變異多肽包含與SEQ ID NO:4的氨基酸序列具有至少90%序列同一性的氨基酸序列，並且LukB蛋白或多肽包含與SEQ ID NO:16的氨基酸序列具有至少90%序列同一性的氨基酸序列。Embodiment 83 is the vaccine composition of embodiment 82, wherein the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and the LukB protein or polypeptide comprises an amino acid sequence identical to that of SEQ ID NO: 4 16 amino acid sequences having at least 90% sequence identity.

實施方案84是實施方案82所述的疫苗組合物，其中LukA變異多肽包含氨基酸序列SEQ ID NO:4，LukB蛋白或多肽包含氨基酸序列SEQ ID NO:16。Embodiment 84 is the vaccine composition of embodiment 82, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:4, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:16.

實施方案85是一種金黃色葡萄球菌疫苗組合物，其包含實施方案33-56中任一項所述的一種或多種變異LukB蛋白或多肽。Embodiment 85 is a S. aureus vaccine composition comprising one or more variant LukB proteins or polypeptides of any one of embodiments 33-56.

實施方案86是實施方案85所述的疫苗組合物，其進一步包含殺白細胞素A (LukA)蛋白或多肽，所述LukA蛋白或多肽與SEQ ID NO:1 (CC8)的氨基酸序列具有至少85%的序列相似性。Embodiment 86 is the vaccine composition of embodiment 85, further comprising a leukocidin A (LukA) protein or polypeptide having at least 85% the amino acid sequence of SEQ ID NO: 1 (CC8) sequence similarity.

實施方案87是實施方案85所述的疫苗組合物，其進一步包含殺白細胞素A (LukA)蛋白或多肽，所述LukA蛋白或多肽與SEQ ID NO:2 (CC45)的氨基酸序列具有至少85%的序列相似性。Embodiment 87 is the vaccine composition of embodiment 85, further comprising a leukocidin A (LukA) protein or polypeptide having at least 85% the amino acid sequence of SEQ ID NO:2 (CC45) sequence similarity.

實施方案88是一種金黃色葡萄球菌疫苗組合物，其包含實施方案1-32中任一項所述的LukA變異多肽和實施方案33-56中任一項所述的LukB變異多肽。Embodiment 88 is a S. aureus vaccine composition comprising the LukA variant polypeptide of any one of embodiments 1-32 and the LukB variant polypeptide of any one of embodiments 33-56.

實施方案89是實施方案74所述的疫苗組合物，其中LukA變異多肽包含與SEQ ID NO:3的氨基酸序列具有至少90%序列同一性的氨基酸序列，並且LukB蛋白或多肽包含與SEQ ID NO:15的氨基酸序列具有至少90%序列同一性的氨基酸序列。Embodiment 89 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, and the LukB protein or polypeptide comprises an amino acid sequence identical to that of SEQ ID NO: 3 15 amino acid sequences having at least 90% sequence identity.

實施方案90是實施方案74所述的疫苗組合物，其中LukA變異多肽包含SEQ ID NO:3的氨基酸序列，LukB蛋白或多肽包含SEQ ID NO:15的氨基酸序列。Embodiment 90 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:15.

實施方案91是實施方案74所述的疫苗組合物，其中LukA變異多肽包含與SEQ ID NO:3的氨基酸序列具有至少90%序列同一性的氨基酸序列，並且LukB蛋白或多肽包含與SEQ ID NO:17的氨基酸序列具有至少90%序列同一性的氨基酸序列。Embodiment 91 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO: 3 The amino acid sequence of 17 is an amino acid sequence having at least 90% sequence identity.

實施方案92是實施方案74所述的疫苗組合物，其中LukA變異多肽包含SEQ ID NO:3的氨基酸序列，LukB蛋白或多肽包含SEQ ID NO:17的氨基酸序列。Embodiment 92 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:17.

實施方案93是實施方案74所述的疫苗組合物，其中LukA變異多肽包含與SEQ ID NO:4的氨基酸序列具有至少90%序列同一性的氨基酸序列，並且LukB蛋白或多肽包含與SEQ ID NO:18的氨基酸序列具有至少90%序列同一性的氨基酸序列。Embodiment 93 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and the LukB protein or polypeptide comprises an amino acid sequence identical to that of SEQ ID NO: 4 The amino acid sequence of 18 has an amino acid sequence of at least 90% sequence identity.

實施方案94是實施方案74所述的疫苗組合物，其中LukA變異多肽包含SEQ ID NO:4的氨基酸序列，LukB蛋白或多肽包含SEQ ID NO:18的氨基酸序列。Embodiment 94 is the vaccine composition of embodiment 74, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:4, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:18.

實施方案95是實施方案69-94中任一項所述的疫苗組合物，其進一步包含佐劑。Embodiment 95 is the vaccine composition of any one of embodiments 69-94, further comprising an adjuvant.

實施方案96是實施方案69-94中任一項所述的疫苗組合物，其進一步包含一種或多種額外的金黃色葡萄球菌抗原。Embodiment 96 is the vaccine composition of any one of embodiments 69-94, further comprising one or more additional S. aureus antigens.

實施方案97是一種在受試者中產生針對金黃色葡萄球菌的免疫應答的方法，所述方法包括:在所述受試者中有效產生針對金黃色葡萄球菌的所述免疫應答的條件下，向所述受試者施用實施方案69-94中任一項所述的疫苗組合物。Embodiment 97 is a method of generating an immune response against S. aureus in a subject, the method comprising: under conditions effective to generate said immune response against S. aureus in said subject, The subject is administered the vaccine composition of any one of embodiments 69-94.

實施方案98是實施方案66-94中任一項所述的疫苗組合物，其中所述疫苗組合物用於在受試者中產生針對金黃色葡萄球菌的免疫應答的方法中。Embodiment 98 is the vaccine composition of any one of embodiments 66-94, wherein the vaccine composition is used in a method of raising an immune response against S. aureus in a subject.

實施例Example

提供以下實施例來說明本發明的實施例，但絕不旨在限制其範圍。The following examples are provided to illustrate embodiments of the invention, but are in no way intended to limit the scope thereof.

實施例Example 1–1- 示例性的Exemplary LukALuka 變異多肽、variant polypeptides, LukBLuk B 變異多肽和穩定的Variant peptides and stable LukABLukAB 複合物Complex

為了表達lukB異二聚體蛋白，將大腸杆菌BL21(DE3)細胞與克隆到pCDFDuet-1中的lukA構建體和克隆到pETDuet-1中的LukB構建體進行共轉化。將轉化體在50µg/ml氨苄青黴素和50µg/ml大觀黴素中培養，以分別在37℃的Luria-Bertani液體培養基中選擇pETDuet-1和pCDFDuet-1，並以190rpm振蕩過夜。為了表達，在37℃下用1:50稀釋的過夜培養物接種新鮮的Terrific Broth液體培養基，以190rpm的速度振蕩，直到培養物達到OD ₆₀₀= 2。然後通過加入異丙基β-d-1-硫代吡喃半乳糖苷誘導表達至最終濃度為1 mM，並在37℃下繼續誘導5小時。在大腸杆菌Origami 2(DE3)細胞的細胞質中表達LukAB異二聚體，包括LukA和/或LukB中的半胱氨酸取代對，以支持二硫鍵的形成。在大腸杆菌BL21(DE3)的周質中LuCA單體的表達是通過在pD861-CH中轉化LuCA構建體來進行的，在37℃下使用最終濃度為4 mM的鼠李糖在Terrific Brot液體培養基(補充有30 g/mL卡那黴素)中誘導4小時。在誘導細胞質和周質表達構建體後，通過在4℃下以4000 rpm離心15分鐘收獲細胞，然後再懸浮在裂解緩沖液中(94% Bugbuster [EMD Millipore]+6% 5M氯化鈉+ 0.4% 4 M咪唑+蛋白酶抑制劑混合物 [ProteaseArrest, G-Biosciences])。在室溫下裂解20分鐘後，裂解物在冰上孵育45分鐘，然後在16100×g、4℃下離心35分鐘。使用AKTA純25M FPLC和HisTrap柱，通過LukA N-末端的6xHis標簽純化蛋白質，並使用咪唑梯度(50-500mM咪唑溶於50 mM磷酸鈉緩沖液，pH 7.4，200 mM氯化鈉)洗脫。通過SDS-PAGE測定的含有純化蛋白質的餾分被合並，並在50mM磷酸鈉緩沖液、pH 7.4、200mM氯化鈉、10%甘油中於4℃透析過夜。純化的蛋白質通過雙肉桂酸(BCA)蛋白質測定法(Pierce)進行定量。 To express the lukB heterodimeric protein, E. coli BL21(DE3) cells were co-transformed with the lukA construct cloned into pCDFDuet-1 and the LukB construct cloned into pETDuet-1. Transformants were cultured in 50 µg/ml ampicillin and 50 µg/ml spectinomycin to select pETDuet-1 and pCDFDuet-1 in Luria-Bertani liquid medium at 37°C, respectively, and shake overnight at 190 rpm. For expression, fresh Terrific Broth broth was inoculated with a 1:50 dilution of the overnight culture at 37°C with shaking at 190 rpm until the culture reached an _OD600 =2. Expression was then induced by adding isopropyl β-d-1-thiogalactopyranoside to a final concentration of 1 mM, and induction was continued for 5 hours at 37°C. LukAB heterodimers, including cysteine substitution pairs in LukA and/or LukB, were expressed in the cytoplasm of E. coli Origami 2(DE3) cells to support disulfide bond formation. Expression of LuCA monomer in the periplasm of Escherichia coli BL21(DE3) was carried out by transforming the LuCA construct in pD861-CH in Terrific Brot broth (supplemented with 30 g/mL kanamycin) for 4 hours. After induction of cytoplasmic and periplasmic expression constructs, cells were harvested by centrifugation at 4000 rpm for 15 min at 4 °C and then resuspended in lysis buffer (94% Bugbuster [EMD Millipore] + 6% 5M NaCl + 0.4 % 4 M imidazole + protease inhibitor cocktail [ProteaseArrest, G-Biosciences]). After 20 min lysis at room temperature, the lysates were incubated on ice for 45 min and then centrifuged at 16,100 × g, 4°C for 35 min. Proteins were purified by a 6xHis tag at the N-terminus of LukA using an AKTA Pure 25M FPLC and HisTrap column, and eluted using an imidazole gradient (50-500 mM imidazole in 50 mM sodium phosphate buffer, pH 7.4, 200 mM sodium chloride). Fractions containing purified protein as determined by SDS-PAGE were pooled and dialyzed overnight at 4°C against 50 mM sodium phosphate buffer, pH 7.4, 200 mM sodium chloride, 10% glycerol. Purified proteins were quantified by biscinnamic acid (BCA) protein assay (Pierce).

表surface 4.4. 本文所述研究中使用的示例性Exemplary LukALuka 、, LukBLuk B 和and LukBLuk B 異二聚體複合物heterodimeric complex 類毒素Toxoid // 毒素名稱Toxin name LukALuka LukALuka 取代replace SEQ ID NO:SEQ ID NO: LukBLuk B LukBLuk B 取代replace SEQ ID NO:SEQ ID NO: RARPR-013 RARPR-013 CC45 W94 CC45 W94 E321A、Lys81Leu、Ser139Ala、Val111Ile、Val191Ile E321A, Lys81Leu, Ser139Ala, Val111Ile, Val191Ile 11 11 CC45 CC45 無 none 16 16 RARPR-015 RARPR-015 CC45 W95 CC45 W95 E321A、Lys81Met、Ser139Ala、Val111Ile、Val191Ile E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile 4 4 CC45 CC45 無 none 16 16 RARPR-017 RARPR-017 CC45 W96 CC45 W96 E321A、Lys81Leu、Ser139Ala、Val111Ile、Val191Ile、Thr247Val E321A, Lys81Leu, Ser139Ala, Val111Ile, Val191Ile, Thr247Val 12 12 CC45 CC45 無 none 16 16 RARPR-019 RARPR-019 CC45 W97 CC45 W97 E321A、Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Thr247Val E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val 8 8 CC45 CC45 無 none 16 16 RARPR-30 RARPR-30 CC45 W95 CC45 W95 E321A、Lys81Met、Ser139Ala、Val111Ile、Val191Ile E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile 4 4 CC45 CC45 Val53Leu Val53Leu the 18 18 RARPR-31 RARPR-31 CC8 W95 CC8 W95 the E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile 3 3 無 none the the RARPR-32 RARPR-32 CC8 W95 CC8 W95 E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile 3 3 CC8 CC8 無 none 15 15 RARPR-33 RARPR-33 CC8 W95 CC8 W95 the E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile 3 3 CC45 CC45 Val53Leu Val53Leu the 18 18 RARPR-34 RARPR-34 CC8 W95 CC8 W95 the E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile 3 3 CC8 CC8 Val53Leu Val53Leu the 17 17 LukA單體 LukA monomer CC8 W97 W72 CC8 W97 W72 E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Thr246Val、Tyr71Cys、Asp137Cys、Gly146Cys、Gly153Cys E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Thr246Val, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys 9 9 無 none the the LukA單體 LukA monomer CC45 W97 W72 CC45 W97 W72 E321A、Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Thr247Val、Tyr72Cys、Asp138Cys、Gly147Cys、Gly154Cys E321A, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys the the 10 10 無 none the the the CC8 W95 W72 CC8 W95 W72 E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Tyr71Cys、Asp137Cys、 Gly146Cys、Gly153Cys E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys 5 5 CC45 CC45 無 none 16 16 the CC8 W95 W72 CC8 W95 W72 E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Tyr71Cys、Asp137Cys、 Gly146Cys、Gly153Cys E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys 5 5 CC45 CC45 Glu45Cys、Thr122Cys、Glu110Cys、Arg155Cys Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys 22 twenty two the CC8 W95 W72 CC8 W95 W72 E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Tyr71Cys、Asp137Cys、 Gly146Cys、Gly153Cys E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys 5 5 CC45 CC45 Val53Leu Val53Leu the 18 18 the CC8 W95 W72 CC8 W95 W72 E320A、Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Tyr71Cys、Asp137Cys、 Gly146Cys、Gly153Cys E320A, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys 5 5 CC45 CC45 Val53Leu、Glu45Cys、Thr122Cys、Glu110Cys、Arg155Cys Val53Leu, Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys the 20 20 CC8Δ10 CC8Δ10 CC8 CC8 C端殘基312-321的缺失 Deletion of C-terminal residues 312-321 64 64 CC8 CC8 無 none 15 15 CC45Δ10 CC45Δ10 CC45 CC45 C端殘基313-322的缺失 Deletion of C-terminal residues 313-322 65 65 CC45 CC45 無 none 16 16 CC8毒素 CC8 toxin CC8 CC8 無 none 1 1 CC8 CC8 無 none 15 15 CC45毒素 CC45 toxin CC45 CC45 無 none 2 2 CC45 CC45 無 none 16 16

實施例Example 2 -2 - 野生型、Wild type, LukALuka 和and LukABLukAB 類毒素的細胞毒性Toxoid Cytotoxicity

通過與野生型LukB毒素比較，使用前單核白血病細胞系THP-1或新分離的原代人多形核白細胞(hPMNs)評估LukB類毒素蛋白(如表4所定義)的細胞毒性。Cytotoxicity of LukB toxoid proteins (as defined in Table 4) was assessed using the pre-monocytic leukemic cell line THP-1 or freshly isolated primary human polymorphonuclear leukocytes (hPMNs) compared to wild-type LukB toxin.

在檢測細胞毒性之前，在佛波醇12-肉豆蔻酸13-乙酸酯的存在下分化THP-1細胞。對於THP-1細胞毒性測定，將包含總共1×105個細胞的50μl RPMI加入96孔板的每個孔中。將LukAB毒素和類毒素蛋白調節至標準蛋白濃度，在冰冷的RPMI培養基中連續稀釋，並將50μl體積的每種蛋白加入合適的孔中。除了僅含RPMI的陰性對照外，將Triton X-100添加到最終濃度為0.1%的陽性對照中。平板在37℃和5% CO2下孵育2小時，然後使用CytoTox-ONE分析(Promega)評估作為膜完整性標志的細胞質酶乳酸脫氫酶的釋放。THP-1 cells were differentiated in the presence of phorbol 12-myristate 13-acetate before assaying for cytotoxicity. For the THP-1 cytotoxicity assay, 50 μl of RPMI containing a total of 1×10 5 cells was added to each well of a 96-well plate. LukAB toxin and toxoid proteins were adjusted to standard protein concentrations, serially diluted in ice-cold RPMI medium, and a volume of 50 μl of each protein was added to appropriate wells. In addition to the negative control containing only RPMI, Triton X-100 was added to the positive control at a final concentration of 0.1%. Plates were incubated at 37°C and 5% CO2 for 2 hours, then the release of the cytoplasmic enzyme lactate dehydrogenase, a marker of membrane integrity, was assessed using the CytoTox-ONE assay (Promega).

LukA和LukAB類毒素對分化的THP-1細胞的細胞毒性列於下表5。分化的THP-1細胞對野生型毒素敏感，因為CC8和CC45 LukAB野生型毒素在低至0.313µg/ml的毒素濃度下殺死了30%或更多的細胞群體。在LukA(Δ10)的C-末端缺失最後10個氨基酸殘基將CC8Δ10毒素的細胞毒性降低到在40 g/mL時細胞死亡低於5%，但是沒有降低CC45Δ10毒素對分化的THP-1細胞的細胞毒性。這兩種LukA單體對分化的THP-1細胞都沒有顯示細胞毒性。這是預期的結果，因為在沒有LukB的情況下，LukA不應形成活性孔複合物。每一種LukAB二聚體類毒素，包括RARPR-33、RARPR-34和RARPR-15，對分化的THP-1細胞顯示出了顯著降低的細胞毒性，在最高測試濃度40 g/mL下，每一種測試類毒素的細胞死亡率為1%或更低。The cytotoxicity of LukA and LukAB toxoids on differentiated THP-1 cells is listed in Table 5 below. Differentiated THP-1 cells were sensitive to wild-type toxins, as CC8 and CC45 LukAB wild-type toxins killed 30% or more of the cell population at toxin concentrations as low as 0.313 µg/ml. Deletion of the last 10 amino acid residues at the C-terminus of LukA(Δ10) reduced the cytotoxicity of CC8Δ10 toxin to less than 5% cell death at 40 g/mL, but did not reduce the effect of CC45Δ10 toxin on differentiated THP-1 cells Cytotoxicity. Neither of these two LukA monomers showed cytotoxicity to differentiated THP-1 cells. This is an expected result, since LukA should not form active pore complexes in the absence of LukB. Each of the LukAB dimer toxoids, including RARPR-33, RARPR-34, and RARPR-15, showed significantly reduced cytotoxicity against differentiated THP-1 cells, and at the highest tested concentration of 40 g/mL, each The cell death rate of the tested toxoid was 1% or less.

表surface 5. LukA5. LukA 或or LukABLukAB 蛋白對分化的protein pair differentiated THP-1THP-1 細胞的細胞毒性，人類單核細胞系，使用標準量的毒素。For cytotoxicity of cells, a human monocytic cell line, standard amounts of toxin were used. 數據以死亡細胞百分比表示。Data are expressed as percentage of dead cells. the LukABLukAB 濃度concentration (µg/mL) (µg/mL) 類毒素Toxoid // 毒素toxin 4040 2020 1010 55 2.52.5 1.251.25 0.6250.625 0.3130.313 0.1560.156 0.0780.078 0.040.04 0.020.02 RARPR-15 RARPR-15 -12 -12 -12 -12 -12 -12 -14 -14 -14 -14 -15 -15 -10 -10 -14 -14 -12 -12 -11 -11 -9 -9 -7 -7 RARPR-30 RARPR-30 0 0 -1 -1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 RARPR-31 RARPR-31 0 0 0 0 -1 -1 0 0 0 0 0 0 -1 -1 -1 -1 0 0 0 0 -1 -1 -1 -1 RARPR-32 RARPR-32 0 0 -1 -1 -1 -1 -1 -1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 RARPR-33 RARPR-33 1 1 1 1 1 1 0 0 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 RARPR-34 RARPR-34 1 1 0 0 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 0 0 0 1 1 CC8 LukA W97 (單體) CC8 LukA W97 (single unit) 1 1 1 1 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 CC45 LukA W97 (單體) CC45 LukA W97 (single unit) 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 2 2 CC8Δ10 CC8Δ10 4 4 3 3 1 1 1 1 0 0 0 0 0 0 0 0 0 0 -1 -1 0 0 0 0 CC45Δ10 CC45Δ10 53 53 39 39 32 32 31 31 33 33 41 41 54 54 55 55 53 53 35 35 13 13 5 5 CC8毒素 CC8 toxin 41 41 44 44 43 43 39 39 39 39 38 38 37 37 30 30 15 15 6 6 2 2 1 1 CC45毒素 CC45 toxin 42 42 34 34 31 31 34 34 40 40 46 46 46 46 36 36 18 18 4 4 1 1 0 0 the the the the the the the the the the the the the

對於hPMNs，在中毒處理前，將所有毒素標準化為2.5 μg/mL(每個亞單位)，然後將20 μl毒素移至96孔板的頂部孔中，並在10μl 1X PBS中連續稀釋2倍。分離多形核白細胞(PMNs)，標準化為每90μl RPMI(10mM HEPES+0.1% HSA)含有200,000個細胞。然後將90μl多形核白細胞移至每個孔中，並將毒素-PMN混合物在37℃和5% CO ₂培養箱中培養1小時。為了評估毒性，使用10μl的CellTiter 96 Aqueous One Solution(CellTiter；Promega)加入到96孔板中，混合物在5% CO ₂中於37℃溫育1.5小時。PMN的生存能力用PerkinElmer EnVision 2103 Multilabel Reader在492nm的吸光度進行評估。 For hPMNs, before intoxication treatment, all toxins were normalized to 2.5 μg/mL (per subunit), then 20 μl of toxins were pipetted into the top well of a 96-well plate and serially diluted 2-fold in 10 μl 1X PBS. Polymorphonuclear leukocytes (PMNs) were isolated and normalized to contain 200,000 cells per 90 μl of RPMI (10 mM HEPES + 0.1% HSA). Then 90 μl of polymorphonuclear leukocytes were pipetted into each well, and the toxin-PMN mixture was incubated in a 37°C and 5% _CO2 incubator for 1 hour. To assess toxicity, 10 μl of CellTiter 96 Aqueous One Solution (CellTiter; Promega) was added to a 96-well plate, and the mixture was incubated for 1.5 hours at 37°C in 5% CO ₂ . The viability of PMNs was assessed by absorbance at 492 nm with a PerkinElmer EnVision 2103 Multilabel Reader.

LukA單體和LukAB二聚體類毒素對人原代PMN細胞的細胞毒性見下表6。在毒素濃度分別為0.313µg/ml和1.25µg/ml時，野生型CC8和CC45毒素對原代人類多形核白細胞的殺滅率超過90%。相比之下，LukB類毒素和LukA單體對這些細胞的細胞毒性顯著降低。CC8 LukA中10個C-末端殘基的缺失基本上消除了對分化的THP-1細胞的細胞毒性，而這種毒素保留了對hPMNs的細胞毒性，在濃度等於或高於5μg/mL時觀察到超過20%的殺滅率。CC8和CC45 LukA單體對hPMNs的細胞毒性很小，正如缺乏對活性孔複合物形成至關重要的LukB組分的類毒素所預期的那樣。與CC8和CC45野生型LukB毒素相比，每種LukB二聚體類毒素對hPMN細胞的細胞毒性顯著降低。RARPR-33 LukAB類毒素以及相關類毒素RARPR-32和-34顯示出比CC8Δ10更低的細胞毒性，RARPR-33在最高測試濃度(20 g/mL)下僅殺死15%的細胞群。The cytotoxicity of LukA monomer and LukAB dimer toxoid to human primary PMN cells is shown in Table 6 below. When the toxin concentrations were 0.313µg/ml and 1.25µg/ml, the wild-type CC8 and CC45 toxins killed more than 90% of primary human polymorphonuclear leukocytes. In contrast, LukB toxoid and LukA monomer were significantly less cytotoxic to these cells. Deletion of 10 C-terminal residues in CC8 LukA essentially abolished cytotoxicity to differentiated THP-1 cells, whereas the toxin retained cytotoxicity to hPMNs, observed at concentrations equal to or higher than 5 μg/mL to more than 20% kill rate. CC8 and CC45 LukA monomers were minimally cytotoxic to hPMNs, as expected for toxoids lacking the LukB component critical for active pore complex formation. Each LukB dimer toxoid was significantly less cytotoxic to hPMN cells than CC8 and CC45 wild-type LukB toxoids. RARPR-33 LukAB toxoid and related toxoids RARPR-32 and -34 showed lower cytotoxicity than CC8Δ10, with RARPR-33 killing only 15% of the cell population at the highest tested concentration (20 g/mL).

表surface 6.6. 使用標準量的毒素測定Toxin Assay Using Standard Amounts LukALuka 或or LukABLukAB 蛋白對人原代多形核細胞的細胞毒性。Cytotoxicity of the protein on primary human polymorphonuclear cells. 數據以死亡細胞百分比表示Data expressed as percentage of dead cells the LukABLukAB 濃度concentration (µg/mL)(µg/mL) 類毒素Toxoid // 毒素toxin 2020 1010 55 2.52.5 1.251.25 0.6250.625 0.3130.313 0.1560.156 0.0780.078 0.040.04 0.020.02 RARPR-15RARPR-15 42 42 36 36 31 31 20 20 14 14 11 11 11 11 11 11 6 6 10 10 11 11 RARPR-30RARPR-30 27 27 17 17 14 14 10 10 5 5 0 0 0 0 0 0 0 0 0 0 0 0 RARPR-31RARPR-31 12 12 2 2 1 1 0 0 0 0 3 3 0 0 0 0 0 0 0 0 0 0 RARPR-32RARPR-32 17 17 6 6 4 4 4 4 2 2 1 1 0 0 0 0 0 0 0 0 0 0 RARPR-33RARPR-33 15 15 8 8 6 6 3 3 1 1 0 0 0 0 0 0 0 0 0 0 0 0 RARPR-34RARPR-34 16 16 9 9 5 5 3 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CC8 LukA W97 (CC8 LukA W97 ( 單體monomer )) 5 5 3 3 2 2 1 1 2 2 1 1 1 1 2 2 0 0 2 2 1 1 CC45 LukA W97 (CC45 LukA W97 ( 單體monomer )) 7 7 6 6 2 2 3 3 4 4 1 1 0 0 1 1 2 2 0 0 0 0 CC8CC8 ΔΔ 1010 29 29 26 26 22 twenty two 18 18 11 11 11 11 4 4 4 4 3 3 4 4 3 3 CC8CC8 毒素toxin 93 93 96 96 97 97 96 96 97 97 93 93 90 90 87 87 75 75 52 52 29 29 CC45CC45 毒素toxin 97 97 97 97 97 97 97 97 96 96 85 85 72 72 57 57 49 49 30 30 19 19

實施例Example 3-RARPR-33 LukAB3-RARPR-33 LukAB 類毒素和Toxoid and WT LukABWT LukAB 毒素的其他變體的細胞毒性。Cytotoxicity of other variants of the toxin.

進行了額外的實驗來評估RARPR-33和WT LukAB毒素與LukA單體的不同變體的細胞毒性和免疫原性。將小鼠用於免疫原性研究。Additional experiments were performed to evaluate the cytotoxicity and immunogenicity of different variants of RARPR-33 and WT LukAB toxins with LukA monomers. Mice were used for immunogenicity studies.

在人類多形核白細胞上評估LukAB毒素、類毒素和單體的細胞毒性。中毒處理前，將所有毒素標準化至100 μg/mL(每亞單位)，然後將20 μl毒素移至96孔板的頂部孔中，並在10μl 1X PBS中連續稀釋2倍。PMN從不同的供體中分離出來，標準化為每90μl RPMI(10mM HEPES+0.1% HSA)含有200,000個細胞。將90μl多形核白細胞移至每個孔中，並將毒素-PMN混合物在37℃+ 5% CO2培養箱中培養1小時。為了評估毒性，使用10μl CellTiter 96 Aqueous One Solution (CellTiter; Promega)加入到96孔板中，混合物在5% CO2中於37℃溫育1.5小時。PMN的生存能力用PerkinElmer EnVision 2103 Multilabel Reader在492nm的吸光度進行評估。死亡細胞的百分比通過減去背景(健康細胞+ PBS)並歸一化為100%死亡的Triton X100處理的細胞來計算。Cytotoxicity of LukAB toxins, toxoids and monomers assessed on human polymorphonuclear leukocytes. Before intoxication treatment, all toxins were normalized to 100 μg/mL (per subunit), then 20 μl of toxins were pipetted into the top well of a 96-well plate and serially diluted 2-fold in 10 μl 1X PBS. PMNs were isolated from various donors and normalized to contain 200,000 cells per 90 μl of RPMI (10 mM HEPES + 0.1% HSA). Pipette 90 μl of polymorphonuclear leukocytes into each well and incubate the toxin-PMN mixture in a 37°C + 5% CO2 incubator for 1 hour. To assess toxicity, 10 μl of CellTiter 96 Aqueous One Solution (CellTiter; Promega) was added to a 96-well plate and the mixture was incubated for 1.5 hours at 37°C in 5% CO2. The viability of PMNs was assessed by absorbance at 492 nm with a PerkinElmer EnVision 2103 Multilabel Reader. The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to 100% dead Triton X100-treated cells.

圖3提供了LukA單體和LukAB毒素對人原代PMN細胞的細胞毒性。野生型LukAB CC8和CC45毒素在毒素濃度分別為2.5µg/ml和5µg/ml時，對原代人類多形核白細胞的殺滅率超過90%。在2.5µg/ml的濃度下，還觀察到LukAB混合毒素CC8/CC45和CC45/CC8的最大殺滅率。相比之下，LukB類毒素和LukA單體對這些細胞的細胞毒性顯著降低。CC8 LukA中10個碳末端殘基的缺失保留了對hPMNs的細胞毒性，在濃度等於或高於5µg/ml時觀察到超過20%的殺滅率。CC8和CC45 LukA單體以及這些單體的組合對hPMNs幾乎沒有細胞毒性。RARPR-33 LukAB類毒素顯示出比CC8Δ10C更低的細胞毒性，在最高測試濃度(20 g/mL)下，RARPR-33僅殺死15%的細胞群。Figure 3 provides the cytotoxicity of LukA monomer and LukAB toxin on human primary PMN cells. The killing rate of wild-type LukAB CC8 and CC45 toxins on primary human polymorphonuclear leukocytes exceeded 90% when the toxin concentrations were 2.5µg/ml and 5µg/ml, respectively. At a concentration of 2.5 µg/ml, the maximum killing rate of LukAB mixed toxins CC8/CC45 and CC45/CC8 was also observed. In contrast, LukB toxoid and LukA monomer were significantly less cytotoxic to these cells. Deletion of 10 carbon-terminal residues in CC8 LukA retained cytotoxicity against hPMNs, with over 20% kill observed at concentrations equal to or higher than 5 µg/ml. CC8 and CC45 LukA monomers, as well as combinations of these monomers, had little cytotoxicity to hPMNs. RARPR-33 LukAB toxoid showed lower cytotoxicity than CC8Δ10C, and at the highest tested concentration (20 g/mL), RARPR-33 only killed 15% of the cell population.

實施例Example 4 -4 - 在小鼠中的免疫原性。Immunogenicity in mice.

為了確定不同LukAB變體的免疫原性，對Envigo Hsd:ND4 (4周齡)小鼠(n = 5/抗原)皮下注射20μg LukAB，所述LukAB溶於50 μl 10%甘油1X TBS與50μl佐劑TiterMax® Gold的混合物中。一組5只小鼠也接受了由等體積的10%甘油1X TBS和TiterMax® Gold組成的模擬免疫。在間隔兩周兩次注射相同的抗原-佐劑混合物後，通過心臟穿刺給小鼠放血並獲得血清。 To determine the immunogenicity of different LukAB variants, Envigo Hsd:ND4 (4-week-old) mice (n = 5/antigen) were injected subcutaneously with 20 μg of LukAB dissolved in 50 μl of 10% glycerol 1X TBS with 50 μl of adjuvant agent TiterMax® Gold mixture. A group of 5 mice also received a mock immunization consisting of an equal volume of 10% glycerol 1X TBS and TiterMax® Gold. After two injections of the same antigen-adjuvant mixture two weeks apart, mice were bled by cardiac puncture and serum was obtained.

為了測定抗LukAB抗體滴度，進行了酶聯免疫吸附試驗。將WT LukAB CC8或CC45在1X PBS中稀釋至2 μg/ml，並在96孔Immulon 2HB板(Thermo Fisher，cat no. 3455)中用100 μl溶液包被，並在4℃孵育過夜。然後用洗滌緩沖液(1X PBS + 0.05%吐溫)洗滌平板3次，然後用200 μl封閉緩沖液(含2.5%牛奶的1X PBS)封閉1小時。產生從1:500血清到封閉緩沖液的五倍系列稀釋，並允許在搖板上孵育1小時。然後再次洗滌平板3次，加入在封閉緩沖液中稀釋1:5,000的小鼠IgG-HRP(Biorad)抗體，並允許在室溫下孵育1小時。用洗滌緩沖液連續洗滌三次，洗去未結合的第二抗體。將升至室溫的TMB (100 μl)加入每個孔中，並覆蓋培養25分鐘。反應完成後，向每個反應孔中加入等量的2N硫酸以停止反應。然後在Envision酶標儀上讀取平板的450nm吸光度。圖4A和圖4B所示的熱力圖顯示了重複測量的平均吸光度值，黑色代表高吸光度和抗體與包被抗原的結合，白色代表低吸光度和無抗體結合。To determine anti-LukAB antibody titers, an enzyme-linked immunosorbent assay was performed. WT LukAB CC8 or CC45 was diluted to 2 μg/ml in 1X PBS and coated with 100 μl of the solution in a 96-well Immulon 2HB plate (Thermo Fisher, cat no. 3455) and incubated overnight at 4°C. Plates were then washed 3 times with wash buffer (1X PBS + 0.05% Tween), and then blocked with 200 μl of blocking buffer (1X PBS with 2.5% milk) for 1 hour. Five-fold serial dilutions from 1:500 serum into blocking buffer were generated and allowed to incubate on a rocker plate for 1 hour. Plates were then washed again 3 times and mouse IgG-HRP (Biorad) antibody diluted 1:5,000 in blocking buffer was added and allowed to incubate for 1 hour at room temperature. Wash three times consecutively with wash buffer to wash away unbound secondary antibody. TMB (100 μl) warmed to room temperature was added to each well and incubated covered for 25 minutes. After the reaction was completed, an equal amount of 2N sulfuric acid was added to each reaction well to stop the reaction. The absorbance of the plate was then read at 450 nm on an Envision microplate reader. The heat maps shown in Figure 4A and Figure 4B show the average absorbance values of repeated measurements, with black representing high absorbance and antibody binding to the coated antigen, and white representing low absorbance and no antibody binding.

RARPR-33誘導了強有力的抗CC8和抗CC45 LukAB IgG抗體滴度(圖4A和圖4B)。RARPR-33免疫誘導了與用CC8 WT毒素、CC8/CC45雜交毒素和CC8Δ10C類毒素免疫相當的抗CC8 IgG反應。由單個CC8 LukA單體誘導的抗CC8 LukAB IgG滴度不如由CC8 LukAB毒素或CC8/CC45雜交毒素、CC45/CC8雜交毒素和RARPR 33雜交抗原誘導的抗體滴度高(圖4A)。RARPR-33 induced robust anti-CC8 and anti-CC45 LukAB IgG antibody titers (Figure 4A and Figure 4B). RARPR-33 immunization induced anti-CC8 IgG responses comparable to immunization with CC8 WT toxin, CC8/CC45 hybrid toxin and CC8Δ10C toxoid. Anti-CC8 LukAB IgG titers induced by a single CC8 LukA monomer were not as high as those induced by CC8 LukAB toxin or CC8/CC45 hybrid toxin, CC45/CC8 hybrid toxin, and RARPR 33 hybrid antigen (Fig. 4A).

RARPR-33免疫小鼠的抗CC45 LukAB滴度高於CC8/CC45 WT雜合抗原誘導的滴度，與CC45 WT抗原誘導的滴度相當。CC8和CC45 LukA單體組合可引起對CC8和CC45 LuKA的抗體滴度(圖4B)。然而，由CC8和CC45 LukA單體組合引發的這些抗CC8和抗CC45 LukB滴度不如由RARPR 33引發的那些高。單個CC45 LukA單體可引發非常高的抗CC45 LukB滴度-類似於CC45/CC8混合物引發的水平，僅略低於RARPR-33或CC45 WT毒素引發的水平。這些結果表明，在RARPR-33免疫後，誘導了對LukAB CC8和CC45的高量級抗體反應。The anti-CC45 LukAB titers of RARPR-33 immunized mice were higher than those induced by CC8/CC45 WT hybrid antigen and comparable to those induced by CC45 WT antigen. Combination of CC8 and CC45 LukA monomers elicited antibody titers against both CC8 and CC45 LuKA (Fig. 4B). However, these anti-CC8 and anti-CC45 LukB titers elicited by the combination of CC8 and CC45 LukA monomers were not as high as those elicited by RARPR 33. A single CC45 LukA monomer elicited very high anti-CC45 LukB titers - levels similar to those elicited by CC45/CC8 mixtures and only slightly lower than those elicited by RARPR-33 or CC45 WT toxins. These results indicate that high magnitude antibody responses to LukAB CC8 and CC45 were induced following RARPR-33 immunization.

實施例Example 5 -5 - 抗體介導的毒素細胞毒性中和Antibody-mediated neutralization of toxin cytotoxicity

抗體介導的毒素細胞毒性的中和應用從如上實施例4所述免疫的小鼠獲得的血清進行評估。將熱滅活的混合血清在PBS中標準化為40%血清，然後將20μl血清移至96孔板的頂部孔中，並在10μl 1X PBS中連續稀釋2倍。在室溫下，將每種LukAB毒素克隆複合物序列變體的LD ₉₀加入平板(10μl/孔)中15分鐘。然後將標準化為每80μl RPMI (10mMHEPES+0.1% HSA)含有200,000個細胞的新鮮分離的人原代多形核白細胞(hPMNs)加入血清-毒素混合物中，並在37℃和5% CO ₂下孵育1小時。為了評估毒性，使用10μl CellTiter 96 Aqueous One Solution(CellTiter; Promega)加入到96孔板中，混合物在5% CO ₂中於37℃溫育1.5小時。PMN的生存能力用PerkinElmer EnVision 2103 Multilabel Reader在492nm的吸光度進行評估。抗體中和數據如圖5所示。 Antibody-mediated neutralization of toxin cytotoxicity was assessed using sera obtained from mice immunized as described in Example 4 above. Heat-inactivated pooled serum was normalized to 40% serum in PBS, then 20 μl of serum was transferred to the top well of a 96-well plate and serially diluted 2-fold in 10 μl 1X PBS. The _LD90 of each LukAB toxin cloning complex sequence variant was added to the plate (10 μl/well) for 15 minutes at room temperature. Freshly isolated primary human polymorphonuclear leukocytes (hPMNs) standardized to contain 200,000 cells per 80 μl RPMI (10mM HEPES + 0.1% HSA) were then added to the serum-toxin mixture and incubated at 37°C and 5% _CO2 1 hour. To assess toxicity, 10 μl of CellTiter 96 Aqueous One Solution (CellTiter; Promega) was added to a 96-well plate, and the mixture was incubated at 37°C in 5% _CO for 1.5 hours. The viability of PMNs was assessed by absorbance at 492 nm with a PerkinElmer EnVision 2103 Multilabel Reader. Antibody neutralization data are shown in Figure 5.

來自用RARPR-33免疫的小鼠的血清顯示出所有抗原中最有效的、廣泛的中和LukAB的能力(圖5)。來自RARPR 33免疫小鼠的血清強烈中和了檢測低至0.25%血清的所有11種LukAB變體的細胞毒性，並且對於大多數LukAB變體也提供了檢測低至0.063-0.125%血清的保護(圖5)。用單個CC8和CC45 LukA單體免疫產生的血清具有高度偏向抗原骨架的LukAB中和能力(圖5)。每次免疫以每種單體20 μg(總蛋白40 μg)施用的CC8 LukA單體與CC45 LukA的組合產生了具有廣泛和有效的LukAB中和能力的血清，在低至0.5%的血清中中和了所有11種測試的LukAB變體(圖5)，然而，這低於用RARPR-33免疫小鼠的血清觀察到的水平。Sera from mice immunized with RARPR-33 showed the most potent, broad neutralizing capacity of LukAB among all antigens (Fig. 5). Sera from RARPR 33-immunized mice strongly neutralized the cytotoxicity of all 11 LukAB variants detected down to 0.25% serum and also provided protection for most LukAB variants down to 0.063-0.125% serum ( Figure 5). Sera generated by immunization with individual CC8 and CC45 LukA monomers had LukAB neutralizing capacity highly biased towards the antigenic backbone (Fig. 5). Combination of CC8 LukA monomers administered at 20 μg of each monomer (40 μg total protein) per immunization with CC45 LukA produced sera with broad and potent LukAB neutralizing capacity in as little as 0.5% of serum and all 11 LukAB variants tested (Fig. 5), however, this was below the level observed with sera from mice immunized with RARPR-33.

結合實施例3-5中給出的數據顯示，加入到RARPR-33的CC8/CC45 LukAB骨架中的減毒和穩定的突變提高了CC8/CC45 WT LukAB混合物的廣泛免疫原性效應(圖4和5)，同時與CC8/CC45 WT LukAB毒素相比，RARPR-33也呈現高度減弱的毒性(圖3)。Combined with the data presented in Examples 3-5, it was shown that attenuating and stabilizing mutations added to the CC8/CC45 LukAB backbone of RARPR-33 improved the broad immunogenic effect of the CC8/CC45 WT LukAB mixture (Figure 4 and 5), while compared with CC8/CC45 WT LukAB toxin, RARPR-33 also exhibited highly attenuated toxicity (Fig. 3).

實施例Example 6 -6 - 抗血清毒素中和Anti-serotoxin neutralization

用從用野生型LukAB、野生型LukB混合物(即CC8 LukA/CC45 LukB和CC45 LukA/CC8 LukB)、LukA單體或LukAB類毒素免疫的小鼠獲得的血清評估抗體介導的毒素細胞毒性中和。將熱滅活的混合血清在PBS中標準化為40%血清，然後將20μl血清移至96孔板的頂部孔中，並在10μl 1X PBS中連續稀釋2倍。然後在室溫下，將每種LukAB毒素克隆複合物序列變體的LD ₉₀加入到含有2%、1%或0.5%血清的平板(10μl/孔)的孔中15分鐘。來自不同供體的新鮮分離的人原代多形核白細胞(hPMNs)標準化為每80μl RPMI (10mMHEPES+0.1% HSA)含有200,000個細胞，然後加入血清-毒素混合物中，在37℃和5% CO ₂下孵育1小時。為了評估毒性，使用10μl CellTiter 96 Aqueous One Solution(CellTiter; Promega)加入到96孔板中，混合物在5% CO ₂中於37℃溫育1.5小時。PMN的生存能力用PerkinElmer EnVision 2103 Multilabel Reader在492nm的吸光度進行評估。抗體中和數據顯示在圖6A (2%抗體血清)、圖6B (1%抗體血清)和圖6C (0.5%抗體血清)的表格中。 Antibody-mediated neutralization of toxin cytotoxicity was assessed using sera obtained from mice immunized with wild-type LukAB, wild-type LukB mixtures (i.e., CC8 LukA/CC45 LukB and CC45 LukA/CC8 LukB), LukA monomer, or LukAB toxoid . Heat-inactivated pooled serum was normalized to 40% serum in PBS, then 20 μl of serum was transferred to the top well of a 96-well plate and serially diluted 2-fold in 10 μl 1X PBS. The _LD90 of each LukAB toxin cloning complex sequence variant was then added to wells of the plate (10 μl/well) containing 2%, 1% or 0.5% serum for 15 min at room temperature. Freshly isolated human primary polymorphonuclear leukocytes (hPMNs) from various donors were normalized to contain 200,000 cells per 80 μl RPMI (10mM HEPES + 0.1% HSA) and added to the serum-toxin mixture at 37°C and 5% CO Incubate at _2° C for 1 hour. To assess toxicity, 10 μl of CellTiter 96 Aqueous One Solution (CellTiter; Promega) was added to a 96-well plate, and the mixture was incubated at 37°C in 5% _CO for 1.5 hours. The viability of PMNs was assessed by absorbance at 492 nm with a PerkinElmer EnVision 2103 Multilabel Reader. Antibody neutralization data are shown in the tables in Figure 6A (2% antibody serum), Figure 6B (1% antibody serum), and Figure 6C (0.5% antibody serum).

用野生型CC8和CC45 LukAB免疫誘導抗體以反映免疫抗原序列組成的模式中和LukAB毒素的天然序列變體。由CC8 LukAB毒素引發的抗體有效地中和了來自CC8、CC1、CC5和其他金黃色葡萄球菌譜系的毒素，但它們不能完全中和來自CC30、CC45或ST22A金黃色葡萄球菌的毒素。同樣，用CC45 LukAB毒素免疫誘導的抗體能有效中和來自CC30、CC45或ST22A金黃色葡萄球菌譜系的毒素，但不能中和來自其他譜系的毒素。Immunization with wild-type CC8 and CC45 LukAB induces antibodies that neutralize native sequence variants of the LukAB toxin in a pattern that reflects the sequence composition of the immunized antigen. Antibodies elicited by the CC8 LukAB toxin efficiently neutralized toxins from CC8, CC1, CC5, and other S. aureus lineages, but they did not completely neutralize toxins from CC30, CC45, or ST22A S. aureus. Likewise, antibodies induced by immunization with CC45 LukAB toxin effectively neutralized toxins from CC30, CC45, or ST22A S. aureus lineages, but not toxins from other lineages.

用非天然雜交LukAB免疫小鼠，無論是CC8 LukA與CC45 LukB的組合還是CC45 LukA與CC8 LukB的組合，與天然存在的二聚體組合相比，都產生了對LukAB序列變體顯示具有更廣泛中和作用的抗體。在非天然雜交二聚體中，CC8 LukA和CC45 LukB顯示出比相反組合稍好的中和譜，這種模式保留在LukA倒數第二個殘基中具有Glu至Ala取代的蛋白質中(E323A)。正如針對野生型毒素誘導的抗體所觀察到的，LukA單體誘導的抗體顯示出指示其序列組成的中和模式。CC8LukA和CC45 LukA單體(RARPR-31+CC45 LukA W97)的組合引發了顯示廣泛中和模式的抗體，但是與二聚體抗原相比，中和的效力降低了，1%或0.5%血清的中和水平降低就證明了這一點。Immunization of mice with a non-naturally hybrid LukAB, either the combination of CC8 LukA and CC45 LukB or the combination of CC45 LukA and CC8 LukB, produced a broad spectrum of LukAB sequence variants compared to naturally occurring dimer combinations. Neutralizing antibodies. In non-native hybrid dimers, CC8 LukA and CC45 LukB showed slightly better neutralization profiles than the opposite combination, a pattern that was preserved in proteins with a Glu to Ala substitution in the penultimate residue of LukA (E323A) . As observed for antibodies induced against the wild-type toxin, antibodies induced by the LukA monomer displayed a neutralization pattern indicative of its sequence composition. The combination of CC8LukA and CC45 LukA monomers (RARPR-31+CC45 LukA W97) elicited antibodies that showed a broad neutralization pattern, but neutralization was less potent compared to the dimeric antigen, with 1% or 0.5% serum This is evidenced by the reduced level of neutralization.

在二聚體類毒素中，RARPR-15、RARPR-33和RARPR-34顯示出對所有測試的LukAB序列變體的廣泛中和抗體反應。非天然野生型二聚體組合也顯示了廣泛的中和譜，盡管中和反應的效力不如觀察到的幾種類毒素。當在2%(圖6A)和1%(圖6B)的血清中測試時，混合野生型和類毒素抗原都顯示出廣泛的中和特性，但是當在0.5%的血清中測試時，對類毒素反應的效力提高是明顯的(圖6C)。在此最低測試濃度下，RARPR-15、RARPR-32、RARPR-33和RARPR-34均顯示出廣泛的中和反應。特別是，RARPR-33引發的血清保留了廣泛的中和反應，而混合野生型抗原和E323A類毒素在0.5%血清時未能引發廣泛的保護反應，並且CC45類毒素RARPR-15在最低測試濃度下引發的中和模式反映了其序列組成，因為僅觀察到CC30、CC45和ST22A LukAB毒素的高水平中和。混合二聚體類毒素RARPR-33引發了一種有效的廣泛中和免疫反應。Among dimeric toxoids, RARPR-15, RARPR-33 and RARPR-34 showed broadly neutralizing antibody responses to all tested LukAB sequence variants. The unnatural wild-type dimer combination also showed a broad neutralization spectrum, although the neutralization was less potent than that observed for several toxoids. Both the mixed wild-type and toxoid antigens showed broad neutralizing properties when tested in 2% (Fig. 6A) and 1% (Fig. Increased potency of the toxin response was evident (Fig. 6C). At this lowest concentration tested, RARPR-15, RARPR-32, RARPR-33, and RARPR-34 all showed broad neutralization. In particular, RARPR-33-primed sera retained a broad neutralizing response, whereas mixed wild-type antigen and E323A toxoid failed to elicit broad protective responses at 0.5% serum, and CC45 toxoid RARPR-15 at the lowest tested concentration The neutralization pattern of the lower priming reflected its sequence composition, as only CC30, CC45 and ST22A high levels of neutralization of LukAB toxins were observed. The mixed dimer toxoid RARPR-33 elicits a potent broadly neutralizing immune response.

實施例Example 7–7– 高濃度下under high concentration RARPR-33RARPR-33 的細胞毒性。cytotoxicity.

方法method ::

細胞毒性試驗 :為了評價每種LukAB蛋白複合物的細胞毒性，用金黃色葡萄球菌毒素對新鮮分離的人原代多形核白細胞(PMNs)進行中毒處理。從不同的供體中分離多形核白細胞，並標準化為每50μl RPMI(10mM HEPES+0.1% HSA)含有200,000個細胞。將PBS中的50 μl毒素添加到細胞中，並將毒素-PMN混合物在37℃的 5% CO ₂培養箱中孵育1小時。為了評估毒性，使用10 μl CellTiter 96 Aqueous One Solution(CellTiter; Promega)加入到96孔板中，混合物在5% CO ₂中於37℃溫育1.5小時。PMN的生存能力用PerkinElmer EnVision 2103 Multilabel Reader在492nm的吸光度進行評估。死亡細胞(%)是通過減去背景(健康細胞+ PBS)並歸一化為設定為100%死亡的經TritonX處理的細胞來計算的。 Cytotoxicity assay : To evaluate the cytotoxicity of each LukAB protein complex, freshly isolated human primary polymorphonuclear leukocytes (PMNs) were intoxicated with S. aureus toxin. Polymorphonuclear leukocytes were isolated from various donors and normalized to contain 200,000 cells per 50 μl RPMI (10 mM HEPES + 0.1% HSA). Add 50 μl of toxin in PBS to the cells and incubate the toxin-PMN mixture in a 5% CO _incubator at 37 °C for 1 h. To assess toxicity, 10 μl of CellTiter 96 Aqueous One Solution (CellTiter; Promega) was added to a 96-well plate, and the mixture was incubated at 37°C in 5% _CO for 1.5 hours. The viability of PMNs was assessed by absorbance at 492 nm with a PerkinElmer EnVision 2103 Multilabel Reader. Dead cells (%) were calculated by subtracting background (healthy cells + PBS) and normalizing to TritonX-treated cells set at 100% dead.

LDH 測定 :為了評估是否每一種相應的LukAB蛋白複合物都能引起細胞溶解，將來自不同供體的新鮮分離的人原代多形核白細胞(PMNs)用金黃色葡萄球菌LukAB毒素進行中毒處理，並測量LDH的釋放。WT毒素在PBS中連續稀釋2倍，並以5-0.0024µg/ml的濃度範圍進行測試。將LukAB類毒素在PBS中稀釋，並以2.5、2、1、1.5和0.5mg/ml進行測試。分離多形核白細胞，並標準化為每50μl RPMI (10mMHEPES+0.1% HSA)含有200,000個細胞。然後將50μl PMNs移至每個孔中，並在每個孔中加入50μl 稀釋的毒素。毒素-PMN混合物在37℃+5% CO ₂培養箱中孵育2小時。為了評估LDH的釋放，平板以1500rpm離心5分鐘，然後從每個孔中取出25μl上清液，並轉移到96孔黑色透明底板中。將25µl CytoTox-ONE均質膜完整性試劑(Promega)加入到黑色透明底96孔板中，並將混合物在室溫下於黑暗中孵育10分鐘。通過記錄激發波長為560nm和發射波長為590nm的熒光，用PerkinElmer EnVision 2103 Multilabel Reader評估細胞裂解。死亡細胞(%)通過減去背景(健康細胞+ PBS)並歸一化為設定為100%死亡的經TritonX處理的細胞來計算。 LDH assay : To assess whether each of the corresponding LukAB protein complexes can cause cytolysis, freshly isolated human primary polymorphonuclear leukocytes (PMNs) from various donors were intoxicated with Staphylococcus aureus LukAB toxin, And measure the release of LDH. WT toxin was serially diluted 2-fold in PBS and tested at a concentration range of 5-0.0024 µg/ml. LukAB toxoid was diluted in PBS and tested at 2.5, 2, 1, 1.5 and 0.5 mg/ml. Polymorphonuclear leukocytes were isolated and normalized to contain 200,000 cells per 50 μl of RPMI (10 mM HEPES + 0.1% HSA). 50 μl of PMNs were then pipetted into each well, and 50 μl of diluted toxin was added to each well. The toxin-PMN mixture was incubated in a 37°C + 5% _CO2 incubator for 2 hours. To assess LDH release, the plates were centrifuged at 1500 rpm for 5 min, after which 25 μl of the supernatant was removed from each well and transferred to a 96-well black clear bottom plate. 25 µl of CytoTox-ONE Homogeneous Membrane Integrity Reagent (Promega) was added to a black clear bottom 96-well plate, and the mixture was incubated at room temperature in the dark for 10 min. Cell lysis was assessed with a PerkinElmer EnVision 2103 Multilabel Reader by recording fluorescence at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Dead cells (%) were calculated by subtracting the background (healthy cells + PBS) and normalizing to TritonX-treated cells set at 100% dead.

結果result ::

在前面的實施例中，LukAB類毒素RARPR-33對hPMNs的細胞毒性被確定達到20µg/ml的濃度。接下來，在存在更高濃度(高達2.5mg/ml)的RARPR-33的情況下，監測人PMN的細胞毒性。在約0.156 g/ml毒素中毒處理1小時後，觀察到基於CellTiter測量的WT LukAB CC8、CC45和CC8/CC45毒素對人PMNs (4-6個供體)的最大細胞毒性(圖7A)。對於RARPR-33和CC8 LukA單體，在濃度為0.5 mg/ml時，用CellTiter測量的死亡細胞百分比約為10%(圖7B)。對濃度高達2.5mg/ml的RARPR-33或CC8 LukA單體的多形核白細胞進行孵育不會進一步增加通過CellTiter測量確定的死亡細胞百分比。In the previous example, the cytotoxicity of LukAB toxoid RARPR-33 on hPMNs was determined up to a concentration of 20 µg/ml. Next, human PMN cytotoxicity was monitored in the presence of higher concentrations (up to 2.5 mg/ml) of RARPR-33. Maximal cytotoxicity of WT LukAB CC8, CC45 and CC8/CC45 toxins to human PMNs (4–6 donors) based on CellTiter measurements was observed after 1 h of toxin intoxication treatment at approximately 0.156 g/ml (Fig. 7A). For RARPR-33 and CC8 LukA monomers, the percentage of dead cells measured with CellTiter was approximately 10% at a concentration of 0.5 mg/ml (Fig. 7B). Incubation of polymorphonuclear leukocytes with RARPR-33 or CC8 LukA monomers at concentrations up to 2.5 mg/ml did not further increase the percentage of dead cells determined by CellTiter measurements.

LD ₁₅值表示誘導15%細胞死亡的抗原濃度。使用線性回歸確定LD ₁₅。對於CC8 WT LukAB，LD ₁₅為0.013µg/ml，對於CC45 WT LukAB，LD ₁₅為0.004µg/ml，對於CC8/CC45LukAB混合物，LD ₁₅為0.002µg/ml。LukAB RARPR-33的半數致死量為2.5mg/ml。通過將RARPR-33的LD ₁₅濃度除以WT抗原的LD ₁₅濃度來比較LD ₁₅值。根據這些觀察，LukAB RARPR-33的毒性比LukAB CC8 WT低 192,308倍以上，比LukAB CC45 WT低 625,000倍以上，比LukAB CC8wt/CC45wt低 1,250,000倍以上。 The LD ₁₅ value represents the antigen concentration that induces 15% cell death. LD ₁₅ was determined using linear regression. LD ₁₅ was 0.013 µg/ml for CC8 WT LukAB, 0.004 _µg /ml for CC45 WT LukAB, and _0.002 µg/ml for CC8/CC45 LukAB mixture. The median lethal dose of LukAB RARPR-33 is 2.5mg/ml. LD ₁₅ values were compared by dividing the LD ₁₅ concentration of RARPR-33 by the LD ₁₅ concentration of WT antigen. Based on these observations, the toxicity of LukAB RARPR-33 was more than 192,308 times lower than that of LukAB CC8 WT, more than 625,000 times lower than that of LukAB CC45 WT, and more than 1,250,000 times lower than that of LukAB CC8wt/CC45wt.

此外，在與不同的WT毒素、CC8 LukA單體或RARPR-33孵育兩小時後，進行LDH測定以評估質膜損傷。暴露於WT毒素、CC8 WT、CC45 WT或CC8/CC45毒素混合物2小時後，誘導人PMN細胞毒性(圖7C)。在毒素濃度為0.625µg/ml時，觀察到由LDH測定的最大細胞死亡。相反，在暴露於濃度高達2.5mg/ml的RARPR-33或CC8 LukA單體兩小時後，沒有觀察到人多形核白細胞的質膜損傷(圖7D)。這些數據表明，在濃度高達2.5mg/ml時，RARPR-33已經脫毒，不能誘導人類多形核白細胞的細胞死亡。與CC8/CC45 WT LukAB毒素相比，結合到RARPR-33的CC8/CC45 LukAB骨架中的高度突變減弱了細胞毒性。In addition, LDH assays were performed to assess plasma membrane damage after two hours of incubation with different WT toxins, CC8 LukA monomers, or RARPR-33. Human PMN cytotoxicity was induced after 2 hours of exposure to WT toxin, CC8 WT, CC45 WT or CC8/CC45 toxin mixture (Fig. 7C). Maximum cell death as measured by LDH was observed at a toxin concentration of 0.625 µg/ml. In contrast, no plasma membrane damage was observed in human polymorphonuclear leukocytes after two hours of exposure to RARPR-33 or CC8 LukA monomers at concentrations up to 2.5 mg/ml (Fig. 7D). These data indicate that at concentrations as high as 2.5 mg/ml, RARPR-33 is detoxified and fails to induce cell death in human polymorphonuclear leukocytes. High mutations in the CC8/CC45 LukAB backbone bound to RARPR-33 attenuated cytotoxicity compared with CC8/CC45 WT LukAB toxins.

實施例Example 8:RARPR-338: RARPR-33 與and D39A/R23ED39A/R23E 類毒素的比較Comparison of Toxoids

產生了基於CC8骨架的LukB類毒素，其中LukA具有D39A突變，LukB具有R23E點突變。這種“D39A/R23E類毒素”在Kailasan, S.等人的“Rational Design of Toxoid Vaccine Candidates for Staphylococcus aureusLeukocidin AB (LukAB),” Toxins 11(6): (2019)中有描述，該文獻在此全文引入作為參考。這種類毒素是在LukAB CC8骨架上產生的，與WT CC8 LukAB毒素相比，毒性減弱了36,000倍以上。用分化為PMN樣的HL-60細胞系測定細胞毒性。在本實驗中，對D39A/R23E類毒素和RARPR-33進行了比較。測定了對人多形核白細胞(PMNs)的細胞毒性，並評估了免疫後誘導廣泛毒素中和抗體的能力。 A LukB toxoid based on the CC8 backbone was generated, with LukA having the D39A mutation and LukB having the R23E point mutation. This "D39A/R23E toxoid" is described in Kailasan, S. et al., "Rational Design of Toxoid Vaccine Candidates for Staphylococcus aureus Leukocidin AB (LukAB)," Toxins 11(6): (2019), available at This entire text is incorporated by reference. This toxoid was generated on the LukAB CC8 backbone and was more than 36,000-fold attenuated in toxicity compared to the WT CC8 LukAB toxin. Cytotoxicity was determined with the HL-60 cell line differentiated into PMN-like. In this experiment, a comparison was made between D39A/R23E toxoid and RARPR-33. Cytotoxicity against human polymorphonuclear leukocytes (PMNs) was measured and the ability to induce broadly toxin-neutralizing antibodies following immunization was assessed.

方法method ::

細胞毒性試驗 :為了評價每種LukAB蛋白複合物的細胞毒性，采用金黃色葡萄球菌毒素對來自不同供體的新鮮分離的人原代多形核白細胞(PMNs)進行中毒處理。分離多形核白細胞，標準化為每50μl RPMI(10mM HEPES+0.1% HSA)含有200,000個細胞。向細胞中加入50μl PBS中的毒素，並將毒素-PMN混合物在37℃的5% CO ₂培養箱中培養2小時。為了評估毒性，將10 μl CellTiter 96 Aqueous One Solution(CellTiter; Promega)加入到96孔板中，混合物在5% CO ₂中於37℃溫育1.5小時。PMN的生存能力用PerkinElmer EnVision 2103 Multilabel Reader在492nm的吸光度進行評估。死亡細胞的百分比通過減去背景(健康細胞+ PBS)並歸一化為100%死亡的經TritonX處理的細胞來計算。 Cytotoxicity assay : To evaluate the cytotoxicity of each LukAB protein complex, freshly isolated human primary polymorphonuclear leukocytes (PMNs) from various donors were intoxicated with S. aureus toxin. Polymorphonuclear leukocytes were isolated and normalized to contain 200,000 cells per 50 μl of RPMI (10 mM HEPES + 0.1% HSA). Add 50 μl of toxin in PBS to the cells and incubate the toxin-PMN mixture in a 5% CO _incubator at 37 °C for 2 h. To assess toxicity, 10 μl of CellTiter 96 Aqueous One Solution (CellTiter; Promega) was added to a 96-well plate, and the mixture was incubated at 37°C in 5% _CO for 1.5 hours. The viability of PMNs was assessed by absorbance at 492 nm with a PerkinElmer EnVision 2103 Multilabel Reader. The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to 100% dead TritonX-treated cells.

LDH 測定 :為了評估每種相應的LukAB蛋白複合物是否引起細胞裂解，采用金黃色葡萄球菌LukAB毒素對來自不同供體的新鮮分離的人原代多形核白細胞(PMNs)進行中毒處理，並測量LDH的釋放。WT毒素在PBS中連續稀釋2倍，並在0.5µg/ml~0.00024µg/ml的濃度範圍內進行測試。將LukAB類毒素在PBS中稀釋至1mg/ml~0.03125mg/ml的濃度，並進行測試。分離多形核白細胞，並標準化為每50μl RPMI (10mMHEPES+0.1% HSA)含有200,000個細胞。然後將PMNs(50μl )移至每個孔中，並在每個孔中加入50μl稀釋的毒素。毒素-PMN混合物在37℃+5% CO ₂培養箱中孵育2小時。為了評估LDH的釋放，平板以1500rpm離心5分鐘，然後從每個孔中取出25μl上清液，並轉移到96孔黑色透明底板中。將25µl CytoTox-ONE均質膜完整性試劑(Promega)加入到黑色透明底96孔板中，並將混合物於室溫下在黑暗中孵育10分鐘。通過記錄激發波長為560nm和發射波長為590nm的熒光，用PerkinElmer EnVision 2103 Multilabel Reader評估細胞裂解。死亡細胞的百分比通過減去背景(健康細胞+ PBS)並歸一化到設定為100%死亡的經TritonX處理的細胞來計算。 LDH assay : To assess whether each respective LukAB protein complex caused cell lysis, freshly isolated human primary polymorphonuclear leukocytes (PMNs) from various donors were intoxicated with S. aureus LukAB toxin and measured Release of LDH. WT toxin was serially diluted 2-fold in PBS and tested at concentrations ranging from 0.5 µg/ml to 0.00024 µg/ml. The LukAB toxoid was diluted in PBS to a concentration of 1 mg/ml~0.03125 mg/ml and tested. Polymorphonuclear leukocytes were isolated and normalized to contain 200,000 cells per 50 μl of RPMI (10 mM HEPES + 0.1% HSA). PMNs (50 μl) were then pipetted into each well, and 50 μl of diluted toxin was added to each well. The toxin-PMN mixture was incubated in a 37°C + 5% _CO2 incubator for 2 hours. To assess LDH release, the plates were centrifuged at 1500 rpm for 5 min, after which 25 μl of the supernatant was removed from each well and transferred to a 96-well black clear bottom plate. 25 µl of CytoTox-ONE Homogeneous Membrane Integrity Reagent (Promega) was added to a black clear bottom 96-well plate, and the mixture was incubated at room temperature in the dark for 10 minutes. Cell lysis was assessed with a PerkinElmer EnVision 2103 Multilabel Reader by recording fluorescence at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to TritonX-treated cells set at 100% dead.

小鼠免疫接種：Envigo Hsd:ND4 (4周齡)小鼠(n = 5/抗原)皮下注射20μg LukAB，所述LukAB溶於50 μl 10%甘油1X TBS與50μl佐劑TiterMax® Gold的混合物中。在兩次加強相同的抗原/佐劑混合物後，通過心臟穿刺給小鼠放血，並獲得血清用於毒素中和研究。 Mouse Immunization: Envigo Hsd:ND4 (4-week-old) mice (n = 5/antigen) were injected subcutaneously with 20 μg of LukAB dissolved in a mixture of 50 μl 10% glycerol 1X TBS and 50 μl adjuvant TiterMax® Gold . After two boosts of the same antigen/adjuvant mixture, mice were bled by cardiac puncture and sera were obtained for toxin neutralization studies.

毒素中和試驗：將每組免疫小鼠的血清混合，並在55 ̊C的水浴中加熱滅活30分鐘。然後用PBS將合並的熱滅活血清稀釋至40%。然後通過在96孔板中將40%儲備液在10μl PBS中連續稀釋2倍來實現血清的進一步稀釋。將毒素(10 μl)加入血清孔中，最終濃度為0.156 μg/ml毒素(LD ₉₀)。在每個孔中加入濃度為200,000個RPMI細胞+ 0.1% HSA + 10 mM HEPES的80μl hPMNs。然後將平板在37℃+5% CO ₂培養箱中孵育1小時。孵育後，將Cell Titer加入中毒中並孵育1.5小時。孵育後，然後在酶標儀上以492 nm的吸光度讀取平板。死亡細胞的百分比通過減去背景(健康細胞+PBS)並歸一化到設定為100%死亡的經TritonX處理的細胞來計算。 Toxin neutralization test: The sera of immunized mice in each group were mixed and heat-inactivated in a water bath at 55 ̊C for 30 minutes. The pooled heat-inactivated sera were then diluted to 40% with PBS. Further dilution of sera was then achieved by serial 2-fold dilution of 40% stock solutions in 10 μl PBS in 96-well plates. Toxin (10 μl) was added to the serum wells to a final concentration of 0.156 μg/ml toxin (LD ₉₀ ). Add 80 μl of hPMNs at a concentration of 200,000 RPMI cells + 0.1% HSA + 10 mM HEPES to each well. The plates were then incubated for 1 hour at 37°C + 5% CO ₂ incubator. After incubation, add Cell Titer to the intoxication and incubate for 1.5 hours. After incubation, the plate was then read at absorbance at 492 nm on a microplate reader. The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to TritonX-treated cells set at 100% dead.

結果result ::

據報道，D39A/R23E類毒素的細胞毒性試驗高達約12µg/ml。在此，測定了濃度高達1mg/ml的RARPR-33和D39A/R23E類毒素對人多形核白細胞的細胞毒性。此外，對WT LukAB CC8、CC45和CC8/CC45進行了對比測試。在約0.02µg/mlWT LukAB CC8/CC45、約0.03µg/mlLukAB CC8和0.125µg/mlLukAB CC45中毒處理1小時後，觀察到基於CellTiter測量的人類多形核白細胞的最大細胞毒性(圖8A)。圖中顯示了5名供體的平均值。對於D39A/R23E類毒素，在與1mg/ml孵育時觀察到約22%的細胞毒性。對於RARPR-33，在1mg/ml的濃度下，用CellTiter測量的死亡細胞百分比約為3%(圖8B)。Cytotoxicity assays of D39A/R23E toxoids have been reported up to approximately 12 µg/ml. Here, the cytotoxicity of RARPR-33 and D39A/R23E toxoids at concentrations up to 1 mg/ml on human polymorphonuclear leukocytes was determined. In addition, WT LukAB CC8, CC45 and CC8/CC45 were tested comparatively. The maximum cytotoxicity of human polymorphonuclear leukocytes based on CellTiter measurements was observed after intoxication treatment with approximately 0.02 µg/ml WT LukAB CC8/CC45, approximately 0.03 µg/ml LukAB CC8 and 0.125 µg/ml LukAB CC45 for 1 hour (Fig. 8A). The graph shows the average of 5 donors. For D39A/R23E toxoid, approximately 22% cytotoxicity was observed upon incubation with 1 mg/ml. For RARPR-33, at a concentration of 1 mg/ml, the percentage of dead cells measured with CellTiter was about 3% (Fig. 8B).

此外，在與不同的WT毒素、D39A/R23E類毒素和RARPR-33孵育兩小時後，進行LDH測定以評估質膜損傷。在暴露於WT毒素、CC8 WT、CC45 WT和CC8/CC45毒素混合物的組合2小時後，誘導人PMN的細胞毒性(圖8C)。在毒素濃度為0.25µg/ml時，觀察到由LDH測定的最大細胞死亡率。在人PMN暴露於濃度高達1mg/ml的D39A/R23E類毒素兩小時後，觀察到約8%的細胞死亡。當用類似濃度的RARPR-33孵育人PMN時，沒有觀察到質膜損傷，表明沒有細胞死亡(圖8D)。這些結果表明，RARPR-33在檢測中被減弱到檢測極限以下，並且比D39A/R23E類毒素更弱。In addition, LDH assays were performed to assess plasma membrane damage after two hours of incubation with different WT toxins, D39A/R23E toxoids and RARPR-33. Human PMN cytotoxicity was induced after 2 hours of exposure to a combination of WT toxin, CC8 WT, CC45 WT and CC8/CC45 toxin mixture ( FIG. 8C ). Maximum cell death as measured by LDH was observed at a toxin concentration of 0.25 µg/ml. Approximately 8% cell death was observed after human PMNs were exposed to D39A/R23E toxoid at concentrations up to 1 mg/ml for two hours. When human PMNs were incubated with similar concentrations of RARPR-33, no plasma membrane damage was observed, indicating no cell death (Fig. 8D). These results indicated that RARPR-33 was attenuated in the assay below the limit of detection and was weaker than the D39A/R23E toxoid.

來自用RARPR-33或D39A/R23E類毒素免疫的小鼠的血清在毒素中和試驗中進行測試，以評估該血清在防止毒素誘導的人多形核白細胞細胞死亡的能力。在從4個供體分離的多形核白細胞上測試了對16種不同LukAB毒素的中和作用。Sera from mice immunized with RARPR-33 or D39A/R23E toxoids were tested in a toxin neutralization assay to assess the ability of the sera to prevent toxin-induced human polymorphonuclear leukocyte cell death. Neutralization of 16 different LukAB toxins was tested on polymorphonuclear leukocytes isolated from 4 donors.

在來自RARPR 33免疫小鼠的0.125%血清的存在下，測試的所有16種LukAB變體的細胞毒性作用被中和(圖9)。在相似的血清濃度下，來自D39A/R23E類毒素免疫小鼠的血清僅與來自RARPR-33免疫小鼠的血清同等地保護免受LukAB CC8的細胞毒性影響。針對所有其他毒素，對來自D39A/R23E類毒素免疫小鼠的血清沒有觀察到保護作用或觀察到低得多的保護作用。這些結果表明，RARPR-33免疫誘導了比用D39A/R23E類毒素免疫更廣泛的毒素中和反應。The cytotoxic effects of all 16 LukAB variants tested were neutralized in the presence of 0.125% serum from RARPR 33 immunized mice ( FIG. 9 ). At similar serum concentrations, sera from D39A/R23E toxoid-immunized mice were only as protective against the cytotoxic effects of LukAB CC8 as sera from RARPR-33-immunized mice. Against all other toxins, no or much lower protection was observed with sera from D39A/R23E toxoid-immunized mice. These results suggest that immunization with RARPR-33 induced a broader toxin-neutralizing response than immunization with D39A/R23E toxoid.

實施例Example 9:LukAB9:LukAB 類毒素的熱穩定性Thermal Stability of Toxoids

與野生型蛋白質相比，LukAB類毒素的穩定性通過熱展開實驗來評估，該實驗使用固有的色氨酸或酪氨酸熒光來估計熔化溫度(Tm)，該溫度對應於蛋白質從折疊狀態向展開狀態轉變的中點。使用NanoTemper's PromethiusNT.Plex儀器(NanoTemper Inc., 德國)來評估熱穩定性。對0.3~1mg/ml的蛋白質樣品(20μl，緩沖液:50mM磷酸鈉緩沖液，200mM氯化鈉，酸鹼度7.4，10%甘油)進行熱展開測量，每個樣品重複進行。Prometheus NanoDSF用戶界面(Melting Scan tab)用於設置運行的實驗參數。典型樣品的熱掃描範圍為20℃~95℃，速率為1.0℃/分鐘。將用於樣品的同一緩沖液中的標準單克隆抗體(CNTO5825或NIST)作為對照，重複進行試驗。使用供應商軟件PR.ThermControl分析熱熔化曲線以測定50%蛋白質展開的溫度(Tm)。The stability of the LukAB toxoid compared to the wild-type protein was assessed by thermal unfolding experiments using intrinsic tryptophan or tyrosine fluorescence to estimate the melting temperature (Tm), which corresponds to the transition of the protein from the folded state to Expand the midpoint of the state transition. Thermal stability was assessed using NanoTemper's Promethius NT.Plex instrument (NanoTemper Inc., Germany). Thermal expansion measurements were performed on protein samples (20 μl, buffer: 50 mM sodium phosphate buffer, 200 mM sodium chloride, pH 7.4, 10% glycerol) at 0.3–1 mg/ml in duplicate for each sample. The Prometheus NanoDSF user interface (Melting Scan tab) is used to set the experimental parameters for the run. The thermal scan range for a typical sample is 20°C to 95°C at a rate of 1.0°C/min. Experiments were repeated using standard monoclonal antibodies (CNTO5825 or NIST) in the same buffer as the samples used as controls. Thermal melting curves were analyzed using the supplier's software PR.ThermControl to determine the temperature (Tm) at which 50% of the protein unfolded.

表7A和7B顯示了通過nanoDSF評估的LukA和LukAB類毒素蛋白的熱穩定性。顯示了蛋白質展開開始的溫度(Tonset)和蛋白質展開轉變的中點(Tm1 )，以及具有和不具有穩定取代的可比構建體之間的Tm差異(ΔTm)。Tables 7A and 7B show the thermal stability of LukA and LukAB toxoid proteins assessed by nanoDSF. The temperature at which protein unfolding begins (Tonset) and the midpoint of the protein unfolding transition (Tm1 ), and the difference in Tm between comparable constructs with and without stabilizing substitutions (ΔTm) are shown.

表 7A.CC45遺傳背景中的單取代和混合CC8/CC45遺傳背景中的組合取代。 毒素名稱 LukA LukB Tonset Tm1 Δ Tm ^a CC45毒素 CC45 LukA ^E321A CC45 LukB ^wt 40.3 47.3 -- CC8/CC45毒素 CC8 LukA ^E320A CC45 LukB ^wt 37.7 43.9 -- CC45 LukA ^E321A ^、 ^Lys81Met CC45 LukB ^wt 40.5 47.4 0.1 CC45 LukA ^E321A ^、 ^Ser139Ala CC45 LukB ^wt 40.5 47.4 0.1 CC45 LukA ^E321A ^、 ^Val111Ile CC45 LukB ^wt 40.0 47.7 0.4 CC45 LukA ^E321A ^、 ^Val190Ile CC45 LukB ^wt 40.5 47.5 0.2 CC45 LukA ^E321A ^、 ^Thr247Val CC45 LukB ^wt 41.3 47.3 0 CC45 LukA ^E321A CC45 LukB ^Val53Leu 40.7 47.8 0.5 RARPR-15 CC45 LukA ^E321A ^、 ^Lys81Met ^、 ^Ser139Ala ^、 ^Val111Ile ^、 ^Val191Ile CC45 LukB ^wt 41.2 48.9 1.6 RARPR-33 CC8 LukA ^E320A ^、 ^Lys80Met ^、 ^Ser138Ala ^、 ^Val110Ile ^、 ^Val190Ile CC45 LukB ^Val53Leu 40.0 47.9 4.0 LukA單體 CC8 LukA ^E320A ^、 ^Lys80Met ^、 ^Ser138Ala ^、 ^Val110Ile ^、 ^Val190Ile ^、 ^Thr246Val ^、 ^Tyr71Cys ^、 ^Asp137Cys ^、 ^Gly146Cys ^、 ^Gly153Cys 無LukB 45.2 61.8 -- LukA單體 CC45 LukA ^E321A ^、 ^Lys81Met ^、 ^Ser139Ala ^、 ^Val111Ile ^、 ^Val191Ile ^、 ^Thr247Val ^、 ^Tyr72Cys ^、 ^Asp138Cys ^、 ^Gly147Cys ^、 ^Gly154Cys 無LukB 51.9 58.1 -- ^aΔTm表示與攜帶一個或多個取代的相應LukAB蛋白相比，沒有穩定取代和二硫鍵的CC45或CC8/CC45毒素的Tm值之間的差異。 LukA單體包括一個N-端PelB信號序列，用於指導表達到大腸杆菌的周質，以支持二硫鍵的形成。攜帶支持二硫鍵形成的成對半胱氨酸取代對的LukAB二聚體在大腸杆菌Origami 2(DE3)細胞的細胞質中表達。 Table 7A. Single substitutions in CC45 genetic background and combined substitutions in mixed CC8/CC45 genetic background. Toxin name Luka Luk B Tonset Tm1 Δ Tm ^a CC45 toxin CC45 LukA ^E321A CC45 LukB ^wt 40.3 47.3 -- CC8/CC45 toxin CC8 LukA ^E320A CC45 LukB ^wt 37.7 43.9 -- CC45 LukA ^E321A ^, ^Lys81Met CC45 LukB ^wt 40.5 47.4 0.1 CC45 LukA ^E321A ^, ^Ser139Ala CC45 LukB ^wt 40.5 47.4 0.1 CC45 LukA ^E321A ^, ^Val111Ile CC45 LukB ^wt 40.0 47.7 0.4 CC45 LukA ^E321A ^, ^Val190Ile CC45 LukB ^wt 40.5 47.5 0.2 CC45 LukA ^E321A ^, ^Thr247Val CC45 LukB ^wt 41.3 47.3 0 CC45 LukA ^E321A CC45 LukB ^Val53Leu 40.7 47.8 0.5 RARPR-15 CC45 LukA ^E321A ^, ^Lys81Met ^, ^Ser139Ala ^, ^Val111Ile ^, ^Val191Ile CC45 LukB ^wt 41.2 48.9 1.6 RARPR-33 CC8 LukA ^E320A ^, ^Lys80Met ^, ^Ser138Ala ^, ^Val110Ile ^, ^Val190Ile CC45 LukB ^Val53Leu 40.0 47.9 4.0 LukA monomer CC8 LukA ^E320A ^, ^Lys80Met ^, ^Ser138Ala ^, ^Val110Ile ^, ^Val190Ile ^, ^Thr246Val ^, ^Tyr71Cys ^, ^Asp137Cys ^, ^Gly146Cys ^, ^Gly153Cys No LukB 45.2 61.8 -- LukA monomer CC45 LukA ^E321A ^, ^Lys81Met ^, ^Ser139Ala ^, ^Val111Ile ^, ^Val191Ile ^, ^Thr247Val ^, ^Tyr72Cys ^, ^Asp138Cys ^, ^Gly147Cys ^, ^Gly154Cys No LukB 51.9 58.1 -- ^a ΔTm represents the difference between the Tm values of CC45 or CC8/CC45 toxins without stabilizing substitutions and disulfide bonds compared to the corresponding LukAB protein carrying one or more substitutions. The LukA monomer includes an N-terminal PelB signal sequence to direct expression into the periplasm of E. coli to support disulfide bond formation. LukAB dimers carrying pairs of cysteine substitutions that support disulfide bond formation were expressed in the cytoplasm of E. coli Origami 2 (DE3) cells.

表 7B.CC8遺傳背景中的單取代和混合物CC8/CC45遺傳背景中的組合取代。 毒素名稱 LukA LukB Tonset Tm1 Δ Tm ^a CC45毒素 CC45 LukA ^E321A CC45 LukB ^wt 37.8 45.3 -- CC8/CC45毒素 CC8 LukA ^E320A CC45 LukB ^wt 35.3 42.2 -- CC8 LukA ^E320A ^、 ^Lys80Met CC45 LukB ^wt 38.8 44.9 2.7 CC8 LukA ^E320A ^、 ^Ser138Ala CC45 LukB ^wt 38.9 44.7 2.5 CC8 LukA ^E320A ^、 ^Val110Ile CC45 LukB ^wt 38.0 44.0 1.8 CC8 LukA ^E320A ^、 ^Val190Ile CC45 LukB ^wt 37.2 43.5 1.3 CC8 LukA ^E320A ^、 ^Thr246Ile CC45 LukB ^wt 36.4 43.1 0.9 CC8 LukA ^E320A ^、 ^Lys80Met ^、 ^Ser138Ala ^、 ^Val110Ile ^、 ^Val190Ile ^、 ^Thr246Val CC45 LukB ^wt 40.5 46.8 4.6 RARPR-33 CC8 LukA ^E320A ^、 ^Lys80Met ^、 ^Ser138Ala ^、 ^Val110Ile ^、 ^Val190Ile CC45 LukB ^Val53Leu 38.8 46.3 4.1 Table 7B. Single substitutions in the CC8 genetic background and combined substitutions in the mixed CC8/CC45 genetic background. Toxin name Luka Luk B Tonset Tm1 Δ Tm ^a CC45 toxin CC45 LukA ^E321A CC45 LukB ^wt 37.8 45.3 -- CC8/CC45 toxin CC8 LukA ^E320A CC45 LukB ^wt 35.3 42.2 -- CC8 LukA ^E320A ^, ^Lys80Met CC45 LukB ^wt 38.8 44.9 2.7 CC8 LukA ^E320A ^, ^Ser138Ala CC45 LukB ^wt 38.9 44.7 2.5 CC8 LukA ^E320A ^, ^Val110Ile CC45 LukB ^wt 38.0 44.0 1.8 CC8 LukA ^E320A ^, ^Val190Ile CC45 LukB ^wt 37.2 43.5 1.3 CC8 LukA ^E320A ^, ^Thr246Ile CC45 LukB ^wt 36.4 43.1 0.9 CC8 LukA ^E320A ^, ^Lys80Met ^, ^Ser138Ala ^, ^Val110Ile ^, ^Val190Ile ^, ^Thr246Val CC45 LukB ^wt 40.5 46.8 4.6 RARPR-33 CC8 LukA ^E320A ^, ^Lys80Met ^, ^Ser138Ala ^, ^Val110Ile ^, ^Val190Ile CC45 LukB ^Val53Leu 38.8 46.3 4.1

結果 :熱穩定性分析(表7A)顯示，CC45 LukA ^E321A/CC45 LukB蛋白的Tm值比CC8 LukA ^E321A/CC45 LukB混合蛋白的Tm值高3℃。CC45 LukA中的單個取代，與CC45 LukB ^wt結合，導致Tm適度增加0~0.4℃。LukB中的Val53Leu取代導致Tm增加0.5℃。由於混合LukB類毒素包括CC8 LukA背景，在CC8 LukA中測試了單個氨基酸取代，並與野生型CC45 LukB結合(表7B)。如CC45 LukA所示，CC8 LukA中的單個取代也使Tm值高於野生型LukA。LukA (RARPR-15)中取代的組合產生的Tm值比CC45 LukA ^E321A/CC45 LukB蛋白高1.6℃，而CC8 LukA取代與LukBVal53Leu (RARPR-33)的組合產生的Tm值比CC8 LukA ^E321A/CC45 LukB混合高4℃。在兩個數據集中觀察到RARPR-33的熱穩定性增加(表7A和7B)。盡管nanoDSF可能會對在低於50℃時展開的蛋白質產生一些可變性，但在4.0℃和4.1℃的數據集上，使用每組中的對照樣品測定的ΔTm值是一致的。LukA單體包括取代的組合和半胱氨酸取代對，並且顯示出≥58℃的升高的Tm值，表明二硫鍵對增加熱穩定性的進一步貢獻。 Results : Thermal stability analysis (Table 7A) showed that the Tm value of the CC45 LukA ^E321A /CC45 LukB protein was 3°C higher than that of the CC8 LukA ^E321A /CC45 LukB mixed protein. A single substitution in CC45 LukA, combined with CC45 LukB ^wt , resulted in a modest increase in Tm of 0~0.4°C. Val53Leu substitution in LukB resulted in a 0.5 °C increase in Tm. Since mixed LukB toxoids include the CC8 LukA background, single amino acid substitutions were tested in CC8 LukA and combined with wild-type CC45 LukB (Table 7B). A single substitution in CC8 LukA also resulted in higher Tm values than wild-type LukA, as shown for CC45 LukA. The combination of substitutions in LukA (RARPR-15) produced a Tm value 1.6°C higher than that of the CC45 LukA ^E321A /CC45 LukB protein, while the combination of the CC8 LukA substitution with LukBVal53Leu (RARPR-33) produced a Tm value higher than that of the CC8 LukA ^E321A /CC45 LukB protein. Mix at 4°C high. Increased thermal stability of RARPR-33 was observed in both datasets (Tables 7A and 7B). The ΔTm values determined using the control samples in each group were consistent across the 4.0°C and 4.1°C datasets, although nanoDSF may introduce some variability in proteins unfolding below 50°C. LukA monomers included combinations of substitutions and pairs of cysteine substitutions, and showed elevated Tm values ≥58°C, suggesting a further contribution of disulfide bonds to increased thermal stability.

實施例Example 1-91-9 的討論discussion ::

本文所述的穩定的LukAB變體異二聚體類毒素具有幾個特征，使得它們非常適合作為金黃色葡萄球菌疫苗抗原候選物。The stable LukAB variant heterodimeric toxoids described herein have several features that make them well suited as S. aureus vaccine antigen candidates.

首先，本文所述的LukA單體和LukAB二聚體類毒素，包括RARPR-30、RARPR-31、RARPR-32、RARPR-33、RARPR-34和RARPR-15，與野生型毒素和其他已知類毒素(即CC8Δ10和CC45Δ10類毒素)相比，顯示出對分化的人THP-1和人多形核白細胞的細胞毒性顯著降低。即使濃度高達2.5mg/ml，RARPR-33仍無細胞毒性，證明其完全衰減。First, the LukA monomer and LukAB dimer toxoids described herein, including RARPR-30, RARPR-31, RARPR-32, RARPR-33, RARPR-34, and RARPR-15, were compared with wild-type toxins and other known Toxoids (ie CC8Δ10 and CC45Δ10 toxoids) showed significantly reduced cytotoxicity against differentiated human THP-1 and human polymorphonuclear leukocytes. Even at concentrations as high as 2.5 mg/ml, RARPR-33 was non-cytotoxic, demonstrating its complete attenuation.

其次，相對於僅含有單個取代的相應類毒素，在LukA和LukB變異體蛋白中引入的取代基的組合顯著增強了異二聚體RARPR複合物的熱穩定性。特別地，LuCA(RARPR-15)中取代的組合產生了比CC45 LukAE321A/CC45 LukB蛋白高1.6℃的Tm值，並且CC8 LukA取代與LukBVal53Leu (RARPR-33)的組合導致了比CC8 LukAE321A/CC45 LukB雜種高4℃的Tm值。Second, the combination of substituents introduced in the LukA and LukB variant proteins significantly enhanced the thermostability of the heterodimeric RARPR complex relative to the corresponding toxoids containing only a single substitution. In particular, the combination of substitutions in LuCA (RARPR-15) produced a Tm value that was 1.6°C higher than that of the CC45 LukAE321A/CC45 LukB protein, and the combination of the CC8 LukA substitution with LukBVal53Leu (RARPR-33) resulted in a higher Tm value than the CC8 LukAE321A/CC45 LukB protein. The hybrid has a higher Tm value at 4°C.

除了減弱的細胞毒性和增強的熱穩定性之外，本文所述的LukAB RARPR類毒素，特別是RARPR-15、RARPR-33和RARPR-34，與野生型CC45和CC8毒素、野生型雜交毒素和類毒素，包括E323A類毒素和D39A/R23E類毒素相比，誘導了相當的或更廣泛的毒素中和反應和更高滴度的中和抗體。In addition to reduced cytotoxicity and enhanced thermostability, the LukAB RARPR toxoids described herein, particularly RARPR-15, RARPR-33 and RARPR-34, hybridize with wild-type CC45 and CC8 toxins, wild-type toxins and Toxoids, including E323A toxoids, induced comparable or broader toxin-neutralizing responses and higher titers of neutralizing antibodies than D39A/R23E toxoids.

總之，減弱的細胞毒性、改善的熱穩定性、強大的免疫原性和廣泛的中和抗體特征使本文所述的LukAB RARPR類毒素成為理想的疫苗抗原候選物。Taken together, attenuated cytotoxicity, improved thermostability, robust immunogenicity, and broad neutralizing antibody profile make the LukAB RARPR toxoid described herein an ideal vaccine antigen candidate.

盡管本文已經詳細描述了優選的實施例，但是對於相關領域的技術人員來說，很明顯，在不脫離本發明的精神的情況下，可以進行各種修改、添加、替換等，因此這些都被認為在所附申請專利範圍限定的本發明的範圍內。Although preferred embodiments have been described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, etc. can be made without departing from the spirit of the invention, and therefore these are considered within the scope of the invention as defined by the appended claims.

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圖1是15個不同的金黃色葡萄球菌LukA氨基酸序列的比對，包括克隆複合物(CC) 8 (SEQ ID NO:1)的LukA；CC45 (SEQ ID NO:2)；CC30(SEQ ID NO:27)的HMPREF 0772 _ 044(TC60)；CC30(SEQ ID NO:36)的SAR2108(MRSA 252)；CC45(SEQ ID NO:34)的SALG _ 02329(A9635)；CC398 (SEQ ID NO:35)的SAPIG 2061(ST398)；CC10(SEQ ID NO:37) 的SATG _ 01930(D139)；CC8(SEQ ID NO:26)的NEWMAN；CC151(SEQ ID NO:32)的SAB1876C(RF122)；CC5(SEQ ID NO:38)的SAV2005(Mu50)；CC5(SEQ ID NO:31)的SA1813(N315)；CC8(SEQ ID NO:33)的SACOL 2006；CC7(SEQ ID NO:29)的HMPRE0776_ 0173 USA 300(TCH 959)；CC72(SEQ ID NO:28)的HMPREF0774_ 2356 TCH 130；和CC1(SEQ ID NO:30)的MW1942(MW2)。通過比對產生的大部分LukA序列的氨基酸序列被表述為SEQ ID NO:25。本文所述的氨基酸取代的位置也在每個LukA序列中得到鑒定。圖2是14個不同的金黃色葡萄球菌LukB氨基酸序列的比對，包括LukB CC8 (SEQ ID NO:15)；CC45 (SEQ ID NO:16)；CC45(SEQ ID NO:40)的A9635；CC30(SEQ ID NO:43)的E1410；CC30(SEQ ID NO:45)的MRSA 252；CC10(SEQ ID NO:42)的D139；CC5(SEQ ID NO:46)的Mu.50；CC239(SEQ ID NO:44)的JKD6008；CC8(SEQ ID NO:41)的COL；CC8(SEQ ID NO:50)的USA 300_ FPR 3757；CC8(SEQ ID NO:51)的NEWMAN；CC151(SEQ ID NO:48)的RF122；CC1(SEQ ID NO:47)的MW2；和CC72(SEQ ID NO:49)的TCH130。通過比對產生的大多數LukB序列的氨基酸序列被表述為SEQ ID NO:39。本文所述的氨基酸取代的位置也在每個LukB序列中得到鑒定。圖3示出了用於免疫的不同LukAB變體的細胞毒性。通過滴定不同LukAB變體對原代人多形核白細胞(PMNs，n=4)進行1小時的中毒處理。用細胞滴度評估細胞活力。數據來自4個供體的平均值±SEM，在兩個獨立的實驗中獲得。圖4A–4B示出了用不同LukAB變體免疫的小鼠中針對LukAB CC8或CC45的抗體滴度。Envigo Hsd：向ND4 (4周齡)小鼠(n = 5/抗原)皮下注射20μg LukAB，所述LukAB溶於50 μl 10%甘油1X TBS中與50μl佐劑TiterMax® Gold混合液。一組5只小鼠也接受了由等體積的10%甘油1X TBS和TiterMax® Gold組成的模擬免疫。在相同抗原佐劑混合物的兩次增強免疫(增強免疫之間間隔2周)之後，通過心臟穿刺對小鼠進行放血並獲得血清。收集來自具有指示性免疫抗原的免疫小鼠的血清，並連續稀釋，以確定CC8 LukAB(圖4A)或CC45 LukAB(圖4B)的抗體滴度。用2μg/ml的CC8或CC45 LukAB塗布平板。熱力圖顯示重複測量的平均吸光度值。圖5提供了用針對各種LukAB毒素的不同LukAB變體進行免疫得到的小鼠的血清的中和曲線。在用指示抗原免疫的小鼠的4-0.031%血清存在的情況下，用0.156 μg/ml (LD90)指示性的LukAB變體對人PMNs (n=4)中毒處理1小時。在使用前，收集具有指示性免疫抗原的免疫小鼠的血清並進行熱滅活。用細胞滴度評估細胞活力。熱力圖顯示了4名供體死亡細胞的平均百分比，黑色代表無細胞死亡，白色代表100%細胞死亡。圖6A-6C示出了在不存在或存在用2%(圖6A)、1%(圖6B)和0.5%(圖6C)來自指示性抗原免疫的小鼠血清的情況下，用LukAB毒素序列變體的LD90中毒後死亡的人多形核白細胞的百分比。數據以死亡細胞的百分比表示。無陰影的細胞代表細胞死亡率最低，最深灰色陰影的細胞代表細胞死亡率最高。圖7A-7D示出了高濃度的LukAB RARPR-33處理沒有產生細胞毒性。將來自健康供體(n=4-6)的新鮮分離的人多形核白細胞與不同濃度的LukAB變體一起孵育1小時。細胞活力通過細胞滴度的吸光度來確定(圖7A和7B)。死亡細胞的百分比是通過減去背景(健康細胞+ PBS)並歸一化到設定為100%死亡的Triton X100處理的細胞來計算的。顯示為平均值±SEM。從健康供體(n=4-6)中分離的人多形核白細胞與不同濃度的LukAB變體一起孵育2小時。通過乳酸脫氫酶(LDH)釋放確定(圖7C和7D)細胞活力。顯示為平均值±SEM。圖8A-8D示出了高濃度的LukAB RARPR-33和D39A/R23E類毒素的作用。來自健康供體(n=5)的新鮮分離的人多形核白細胞與不同濃度的LukAB變體一起孵育2小時。細胞活力通過細胞滴度的吸光度來確定(圖8A和8B)。死亡細胞的百分比是通過減去背景(健康細胞+ PBS)並歸一化到設定為100%死亡的Triton X100處理的細胞來計算的。顯示為平均值±SEM。從健康供體(n=5)中分離的人多形核白細胞與不同濃度的LukAB變體一起孵育2小時。通過LDH釋放確定(圖8C和8D)細胞活力。顯示為平均值±SEM。圖9示出了用兩種不同LukAB類毒素進行免疫得到的小鼠的血清對各種LukAB毒素的中和曲線。在用兩種不同抗原免疫的小鼠的0.125%血清存在的情況下，用0.156 μg/ml (LD90)指示性的LukAB毒素變體對人PMNs (n=4)進行中毒處理1小時。收集用兩種指示性免疫抗原進行免疫得到的小鼠的血清並在使用前進行熱滅活。用細胞滴度測定細胞活力。條形圖顯示了4種不同供體的平均值±SEM。使用非配對t檢驗確定統計顯著性，P＜0.05被認為是顯著的。*P＜0.05，**P＜0.001，***P＜0.0001。 Figure 1 is an alignment of 15 different Staphylococcus aureus LukA amino acid sequences, including LukA of Cloning Complex (CC) 8 (SEQ ID NO:1); CC45 (SEQ ID NO:2); CC30 (SEQ ID NO SAR2108 (MRSA 252) of CC30 (SEQ ID NO:36); SALG_02329 (A9635) of CC45 (SEQ ID NO:34); CC398 (SEQ ID NO:35 ) of SAPIG 2061 (ST398); CC10 (SEQ ID NO:37) of SATG_01930 (D139); CC8 (SEQ ID NO:26) of NEWMAN; CC151 (SEQ ID NO:32) of SAB1876C (RF122); CC5 SAV2005 (Mu50) of (SEQ ID NO:38); SA1813 (N315) of CC5 (SEQ ID NO:31); SACOL 2006 of CC8 (SEQ ID NO:33); HMPRE0776_0173 of CC7 (SEQ ID NO:29) USA 300 (TCH 959); HMPREF0774_2356 TCH 130 of CC72 (SEQ ID NO:28); and MW1942 (MW2) of CC1 (SEQ ID NO:30). The amino acid sequence of most of the LukA sequences generated by the alignment is represented as SEQ ID NO:25. The positions of the amino acid substitutions described herein were also identified in each LukA sequence. Figure 2 is an alignment of 14 different Staphylococcus aureus LukB amino acid sequences, including LukB CC8 (SEQ ID NO:15); CC45 (SEQ ID NO:16); A9635 of CC45 (SEQ ID NO:40); CC30 E1410 of (SEQ ID NO:43); MRSA 252 of CC30 (SEQ ID NO:45); D139 of CC10 (SEQ ID NO:42); Mu.50 of CC5 (SEQ ID NO:46); NO:44) JKD6008; CC8 (SEQ ID NO:41) COL; CC8 (SEQ ID NO:50) USA 300_FPR 3757; CC8 (SEQ ID NO:51) NEWMAN; CC151 (SEQ ID NO:48 ) for RF122; CC1 (SEQ ID NO:47) for MW2; and CC72 (SEQ ID NO:49) for TCH130. The amino acid sequence of most of the LukB sequences generated by the alignment is represented as SEQ ID NO:39. The positions of the amino acid substitutions described herein were also identified in each LukB sequence. Figure 3 shows the cytotoxicity of different LukAB variants used for immunization. Primary human polymorphonuclear leukocytes (PMNs, n=4) were intoxicated for 1 h by titrating different LukAB variants. Cell viability was assessed by cell titer. Data are mean±SEM from 4 donors, obtained in two independent experiments. Figures 4A-4B show antibody titers against LukAB CC8 or CC45 in mice immunized with different LukAB variants. Envigo Hsd: ND4 (4 week old) mice (n = 5/antigen) were injected subcutaneously with 20 μg LukAB dissolved in 50 μl 10% glycerol 1X TBS mixed with 50 μl adjuvant TiterMax® Gold. A group of 5 mice also received a mock immunization consisting of an equal volume of 10% glycerol 1X TBS and TiterMax® Gold. After two boosts of the same antigen-adjuvant mixture (2 weeks between boosts), mice were bled by cardiac puncture and sera were obtained. Sera from immunized mice with the indicated immunizing antigens were collected and serially diluted to determine antibody titers to CC8 LukAB (Figure 4A) or CC45 LukAB (Figure 4B). Plates were coated with 2 μg/ml of CC8 or CC45 LukAB. The heatmap shows the average absorbance value of replicate measurements. Figure 5 provides neutralization curves for sera from mice immunized with different LukAB variants against various LukAB toxins. Human PMNs (n=4) were intoxicated with 0.156 μg/ml (LD90) of the indicated LukAB variants for 1 h in the presence of 4-0.031% serum from mice immunized with the indicated antigens. Sera from immunized mice with the indicated immunizing antigens were collected and heat-inactivated prior to use. Cell viability was assessed by cell titer. The heat map shows the average percentage of dead cells for the 4 donors, with black representing no cell death and white representing 100% cell death. Figures 6A-6C show the presence or absence of LukAB toxin sequences with 2% (Figure 6A), 1% (Figure 6B) and 0.5% (Figure 6C) of serum from mice immunized with the indicated antigens Percentage of dead human polymorphonuclear leukocytes after intoxication with variant LD90. Data are expressed as percentage of dead cells. Cells with no shading represent the lowest cell death rate, and cells with the darkest shade of gray represent the highest cell death rate. Figures 7A-7D show that high concentrations of LukAB RARPR-33 treatment did not produce cytotoxicity. Freshly isolated human polymorphonuclear leukocytes from healthy donors (n=4-6) were incubated with various concentrations of LukAB variants for 1 hour. Cell viability was determined by absorbance of cell titers (Figures 7A and 7B). The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to Triton X100-treated cells set at 100% dead. Shown as mean ± SEM. Human polymorphonuclear leukocytes isolated from healthy donors (n = 4-6) were incubated with various concentrations of LukAB variants for 2 hours. Cell viability was determined by lactate dehydrogenase (LDH) release (Figure 7C and 7D). Shown as mean ± SEM. Figures 8A-8D show the effect of high concentrations of LukAB RARPR-33 and D39A/R23E toxoids. Freshly isolated human polymorphonuclear leukocytes from healthy donors (n=5) were incubated with various concentrations of LukAB variants for 2 hours. Cell viability was determined by absorbance of cell titers (Figures 8A and 8B). The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to Triton X100-treated cells set at 100% dead. Shown as mean ± SEM. Human polymorphonuclear leukocytes isolated from healthy donors (n = 5) were incubated with various concentrations of LukAB variants for 2 hours. Cell viability was determined by LDH release (Fig. 8C and 8D). Shown as mean ± SEM. Figure 9 shows the neutralization curves of the serum of mice immunized with two different LukAB toxoids against various LukAB toxins. Human PMNs (n=4) were intoxicated with 0.156 μg/ml (LD90) of the indicated LukAB toxin variants for 1 h in the presence of 0.125% serum from mice immunized with two different antigens. Sera from mice immunized with the two indicated immunizing antigens were collected and heat inactivated prior to use. Cell viability was determined by cell titer. Bar graphs show mean ± SEM for 4 different donors. Statistical significance was determined using an unpaired t-test, with P<0.05 considered significant. *P<0.05, **P<0.001, ***P<0.0001.

Claims

一種SEQ ID NO:25變異的金黃色葡萄球菌殺白細胞素A (LukA)多肽，所述LukA變異多肽包含: 對應於SEQ ID NO:25的氨基酸殘基Lys83、Ser141、Val113和Val193的一個或多個氨基酸殘基上的氨基酸取代。 A mutant Staphylococcus aureus leukocidin A (LukA) polypeptide of SEQ ID NO: 25, the LukA variant polypeptide comprising: Amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113 and Val193 of SEQ ID NO:25.

如請求項1所述的LukA變異多肽，其中所述LukA變異多肽進一步包含: 對應於SEQ ID NO:25的Glu323的氨基酸殘基上的氨基酸取代。 The LukA variant polypeptide as described in claim 1, wherein the LukA variant polypeptide further comprises: Amino acid substitutions at amino acid residues corresponding to Glu323 of SEQ ID NO:25.

如請求項2所述的LukA變異多肽，其中對應於Glu323的氨基酸殘基上的氨基酸取代包括穀氨酸到丙氨酸(Glu323Ala)的取代。The LukA variant polypeptide according to claim 2, wherein the amino acid substitution on the amino acid residue corresponding to Glu323 comprises a substitution of glutamic acid to alanine (Glu323Ala).

如請求項1~3中任一項所述的LukA變異多肽，其中對應於Lys83的氨基酸殘基上的氨基酸取代包括賴氨酸到甲硫氨酸(Lys83Met)的取代。The LukA variant polypeptide according to any one of claims 1 to 3, wherein the amino acid substitution on the amino acid residue corresponding to Lys83 comprises a substitution of lysine to methionine (Lys83Met).

如請求項1~4中任一項所述的LukA變異多肽，其中對應於Ser141的氨基酸殘基上的氨基酸取代包括絲氨酸到丙氨酸(Ser141Ala)的取代。The LukA variant polypeptide according to any one of claims 1 to 4, wherein the amino acid substitution on the amino acid residue corresponding to Ser141 comprises a substitution from serine to alanine (Ser141Ala).

如請求項1~5中任一項所述的LukA變異多肽，其中對應於Val113的氨基酸殘基上的氨基酸取代包括纈氨酸到異亮氨酸(Val113Ile)的取代。The LukA variant polypeptide according to any one of claims 1 to 5, wherein the amino acid substitution on the amino acid residue corresponding to Val113 comprises a substitution from valine to isoleucine (Val113Ile).

如請求項1~6中任一項所述的LukA變異多肽，其中對應於Val193的氨基酸殘基上的氨基酸取代包括纈氨酸到異亮氨酸(Val193Ile)的取代。The LukA variant polypeptide according to any one of claims 1 to 6, wherein the amino acid substitution on the amino acid residue corresponding to Val193 comprises a substitution from valine to isoleucine (Val193Ile).

如請求項2~7中任一項所述的LukA變異多肽，其中所述LukA變異多肽包含對應於SEQ ID NO:25的氨基酸殘基Lys83、Ser141、Val113、Val193和Glu323的每個氨基酸殘基上的氨基酸取代。The LukA variant polypeptide according to any one of claims 2 to 7, wherein the LukA variant polypeptide comprises each amino acid residue corresponding to amino acid residues Lys83, Ser141, Val113, Val193 and Glu323 of SEQ ID NO:25 amino acid substitutions.

如請求項8所述的LukA變異多肽，其中所述氨基酸取代包括Lys83Met、Ser141Ala、Val113Ile、Val193Ile和Glu323Ala。The LukA variant polypeptide according to claim 8, wherein the amino acid substitutions include Lys83Met, Ser141Ala, Val113Ile, Val193Ile and Glu323Ala.

如請求項1所述的LukA變異多肽，其中所述變體是SEQ ID NO:1的CC8LukA變體，其包含對應於SEQ ID NO:1中的Lys80Met、Ser138Ala、Val110Ile、Val190Ile和Glu320Ala的氨基酸取代。The LukA variant polypeptide as claimed in claim 1, wherein the variant is the CC8LukA variant of SEQ ID NO:1, which comprises amino acid substitutions corresponding to Lys80Met, Ser138Ala, Val110Ile, Val190Ile and Glu320Ala in SEQ ID NO:1 .

如請求項10所述的LukA變異多肽，其中所述LukA變異多肽包含SEQ ID NO:3的氨基酸序列。The LukA variant polypeptide according to claim 10, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3.

如請求項1所述的LukA變異多肽，其中所述變體是SEQ ID NO:2的CC45 LukA變體，其包含對應於SEQ ID NO:2中Lys81Met、Ser139Ala、Val111Ile、Val191Ile和Glu321Ala的氨基酸取代。The LukA variant polypeptide as claimed in claim 1, wherein the variant is the CC45 LukA variant of SEQ ID NO:2, which comprises amino acid substitutions corresponding to Lys81Met, Ser139Ala, Val111Ile, Val191Ile and Glu321Ala in SEQ ID NO:2 .

如請求項12所述的LukA變異多肽，其中所述LukA變異多肽包含SEQ ID NO:4的氨基酸序列。The LukA variant polypeptide according to claim 12, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:4.

如請求項1~13中任一項所述的LukA變異多肽，其中所述LukA變異多肽進一步包含: 對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的一個或多個氨基酸殘基上的氨基酸取代。 The LukA variant polypeptide as described in any one of claim items 1 to 13, wherein the LukA variant polypeptide further comprises: Amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Tyr74, Aspl40, Gly149 and Gly156 of SEQ ID NO:25.

如請求項14所述的LukA變異多肽，其中對應於Tyr74的氨基酸殘基上的氨基酸取代包括酪氨酸至半胱氨酸(Tyr74Cys)的取代。The LukA variant polypeptide according to claim 14, wherein the amino acid substitution on the amino acid residue corresponding to Tyr74 comprises a substitution from tyrosine to cysteine (Tyr74Cys).

如請求項14或15所述的LukA變異多肽，其中對應於Asp140的氨基酸殘基上的氨基酸取代包括天冬醯胺到半胱氨酸(Asp140Cys)的取代。The LukA variant polypeptide according to claim 14 or 15, wherein the amino acid substitution on the amino acid residue corresponding to Asp140 comprises a substitution from asparagine to cysteine (Asp140Cys).

如請求項14~16中任一項所述的LukA變異多肽，其中對應於Gly149的氨基酸殘基上的氨基酸取代包括甘氨酸到半胱氨酸(Gly149Cys)的取代。The LukA variant polypeptide according to any one of claims 14 to 16, wherein the amino acid substitution on the amino acid residue corresponding to Gly149 comprises a substitution of glycine to cysteine (Gly149Cys).

如請求項14~17中任一項所述的LukA變異多肽，其中在對應於Gly156的氨基酸殘基上的氨基酸取代包括甘氨酸到半胱氨酸(Gly156Cys)的取代。The LukA variant polypeptide according to any one of claims 14 to 17, wherein the amino acid substitution at the amino acid residue corresponding to Gly156 comprises a substitution of glycine to cysteine (Gly156Cys).

如請求項14~18中任一項所述的LukA變異多肽，其中所述LukA變異多肽包含對應於SEQ ID NO:25的氨基酸殘基Tyr74、Asp140、Gly149和Gly156的每個氨基酸殘基上的氨基酸取代。The LukA variant polypeptide according to any one of claims 14 to 18, wherein the LukA variant polypeptide comprises amino acid residues corresponding to each of amino acid residues Tyr74, Asp140, Gly149 and Gly156 of SEQ ID NO:25 Amino acid substitutions.

如請求項14所述的LukA變異多肽，其中所述變體是SEQ ID NO:1的CC8 LukA變體，包含對應於SEQ ID NO:1的Lys80Met、Ser138Ala、Val110Ile、Val190Ile、Glu320Ala、Tyr71Cys、Asp137Cys、Gly146Cys和Gly153Cys的氨基酸取代。The LukA variant polypeptide as described in claim item 14, wherein said variant is the CC8 LukA variant of SEQ ID NO:1, comprising Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Glu320Ala, Tyr71Cys, Asp137Cys corresponding to SEQ ID NO:1 , Gly146Cys and Gly153Cys amino acid substitutions.

如請求項20所述的LukA變異多肽，其中所述LukA變異多肽包含SEQ ID NO:5的氨基酸序列。The LukA variant polypeptide according to claim 20, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:5.

如請求項14所述的LukA變異多肽，其中所述變體是SEQ ID NO:2的CC45 LukA變體，包含對應於SEQ ID NO:2的Lys81Met、Ser139Ala、Val111Ile、Val191Ile、Glu321Ala、Tyr72Cys、Asp138Cys、Gly147Cys和Gly154Cys的氨基酸取代。The LukA variant polypeptide as described in claim item 14, wherein said variant is the CC45 LukA variant of SEQ ID NO:2, comprising Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Glu321Ala, Tyr72Cys, Asp138Cys corresponding to SEQ ID NO:2 , Gly147Cys and Gly154Cys amino acid substitutions.

如請求項22所述的LukA變異多肽，其中所述LukA變異多肽包含SEQ ID NO:6的氨基酸序列。The LukA variant polypeptide according to claim 22, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:6.

如請求項1~23中任一項所述的LukA變異多肽，其中所述LukA變異多肽進一步包含: 對應於SEQ ID NO:25的氨基酸殘基Thr249的氨基酸殘基上的氨基酸取代。 The LukA variant polypeptide as described in any one of claims 1 to 23, wherein the LukA variant polypeptide further comprises: Amino acid substitution at the amino acid residue corresponding to amino acid residue Thr249 of SEQ ID NO:25.

如請求項24所述的LukA變異多肽，其中對應於Thr249的氨基酸殘基上的氨基酸取代包括蘇氨酸到纈氨酸(Thr249Val)的取代。The LukA variant polypeptide according to claim 24, wherein the amino acid substitution on the amino acid residue corresponding to Thr249 comprises a threonine to valine (Thr249Val) substitution.

如請求項25所述的LukA變異多肽，其中所述變異LukA蛋白包含SEQ ID NO:7或SEQ ID NO:8的氨基酸序列。The LukA variant polypeptide according to claim 25, wherein the variant LukA protein comprises the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

如請求項1~26中任一項所述的LukA變異多肽，還包含:氨基末端信號序列。The LukA variant polypeptide according to any one of claims 1-26, further comprising: an amino-terminal signal sequence.

如請求項27所述的LukA變異多肽，其中所述氨基末端信號序列包含SEQ ID NO:23的氨基酸序列。The LukA variant polypeptide as described in claim 27, wherein the amino-terminal signal sequence comprises the amino acid sequence of SEQ ID NO:23.

如請求項1~28中任一項所述的LukA變異多肽，還包含:氨基末端純化序列。The LukA variant polypeptide according to any one of claims 1-28, further comprising: an amino-terminal purification sequence.

一種編碼請求項1~29中任一項所述的LukA變異多肽的核酸分子。A nucleic acid molecule encoding the LukA variant polypeptide described in any one of Claims 1-29.

一種包含請求項30所述的核酸分子的表達載體。An expression vector comprising the nucleic acid molecule described in claim 30.

一種包含請求項31所述的表達載體的宿主細胞。A host cell comprising the expression vector described in Claim 31.

一種SEQ ID NO:39變異的金黃色葡萄球菌殺白細胞素B(LukB)蛋白或多肽，所述LukB變異多肽包含: 對應於SEQ ID NO:39的氨基酸殘基Val53的氨基酸殘基上的氨基酸取代。 A mutated Staphylococcus aureus leukocidin B (LukB) protein or polypeptide of SEQ ID NO: 39, the LukB mutated polypeptide comprising: Amino acid substitution at the amino acid residue corresponding to amino acid residue Val53 of SEQ ID NO:39.

如請求項33所述的LukB變異多肽，其中對應於Val53的氨基酸殘基上的氨基酸取代包括纈氨酸到亮氨酸(Val53Leu)的取代。The LukB variant polypeptide according to claim 33, wherein the amino acid substitution on the amino acid residue corresponding to Val53 comprises a substitution from valine to leucine (Val53Leu).

如請求項33所述的LukB變異多肽，其中所述變體是SEQ ID NO:15的CC8 LukB變體，其包含對應於SEQ ID NO:15的Val53Leu的氨基酸取代。The LukB variant polypeptide according to claim 33, wherein the variant is the CC8 LukB variant of SEQ ID NO:15, which comprises an amino acid substitution corresponding to Val53Leu of SEQ ID NO:15.

如請求項35所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:17的氨基酸序列。The LukB variant polypeptide as described in claim 35, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:17.

如請求項33所述的LukB變異多肽，其中所述變體是SEQ ID NO:16的CC45 LukB變體，包含對應於SEQ ID NO:16的Val53Leu的氨基酸取代。The LukB variant polypeptide according to claim 33, wherein the variant is the CC45 LukB variant of SEQ ID NO:16, comprising an amino acid substitution corresponding to Val53Leu of SEQ ID NO:16.

如請求項37所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:18的氨基酸序列。The LukB variant polypeptide as described in claim 37, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:18.

如請求項33或34所述的LukB變異多肽，其中所述變體進一步包括對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的一個或多個氨基酸殘基上的氨基酸取代。The LukB variant polypeptide as described in claim 33 or 34, wherein said variant further comprises amino acid substitutions on one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39 .

如請求項39所述的LukB變異多肽，其中對應於Glu45的氨基酸殘基上的氨基酸取代包括穀氨酸到半胱氨酸(Glu45Cys)的取代。The LukB variant polypeptide according to claim 39, wherein the amino acid substitution on the amino acid residue corresponding to Glu45 comprises a substitution of glutamic acid to cysteine (Glu45Cys).

如請求項39或40所述的LukB變異多肽，其中對應於Glu109的氨基酸殘基上的氨基酸取代包括穀氨酸到半胱氨酸(Glu109Cys)的取代。The LukB variant polypeptide according to claim 39 or 40, wherein the amino acid substitution on the amino acid residue corresponding to Glu109 comprises a substitution of glutamic acid to cysteine (Glu109Cys).

如請求項39~41中任一項所述的LukB變異多肽，其中對應於Thr121的氨基酸殘基上的氨基酸取代包括蘇氨酸到半胱氨酸(Thr121Cys)的取代。The LukB variant polypeptide according to any one of claims 39-41, wherein the amino acid substitution on the amino acid residue corresponding to Thr121 comprises a threonine to cysteine (Thr121Cys) substitution.

如請求項39~42中任一項所述的LukB變異多肽，其中對應於Arg154的氨基酸殘基上的氨基酸取代包括精氨酸到半胱氨酸(Arg154Cys)的取代。The LukB variant polypeptide according to any one of claims 39-42, wherein the amino acid substitution on the amino acid residue corresponding to Arg154 comprises arginine to cysteine (Arg154Cys) substitution.

如請求項39~43中任一項所述的LukB變異多肽，其中所述LukB變異多肽包含對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上的氨基酸取代。The LukB variant polypeptide according to any one of claims 39 to 43, wherein the LukB variant polypeptide comprises amino acid residues corresponding to Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39 on each amino acid residue Amino acid substitutions.

如請求項39所述的LukB變異多肽，其中所述變體是SEQ ID NO:15的CC8 LukB變體，包含對應於SEQ ID NO:15的Val53Leu、Glu45Cys、Glu109Cys、Thr121Cys和Arg154Cys的氨基酸取代。The LukB variant polypeptide according to claim 39, wherein the variant is the CC8 LukB variant of SEQ ID NO:15, comprising amino acid substitutions corresponding to Val53Leu, Glu45Cys, Glu109Cys, Thr121Cys and Arg154Cys of SEQ ID NO:15.

如請求項45所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:19的氨基酸序列。The LukB variant polypeptide as described in claim 45, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:19.

如請求項33所述的LukB變異多肽，其中所述變體是SEQ ID NO:16的CC45 LukB變體，包含對應於SEQ ID NO:16的Val53Leu、Glu45Cys、Glu110Cys、Thr123Cys和Arg155Cys的氨基酸取代。The LukB variant polypeptide as claimed in claim 33, wherein the variant is the CC45 LukB variant of SEQ ID NO:16, comprising amino acid substitutions corresponding to Val53Leu, Glu45Cys, Glu110Cys, Thr123Cys and Arg155Cys of SEQ ID NO:16.

如請求項47所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:20的氨基酸序列。The LukB variant polypeptide as described in claim 47, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:20.

一種SEQ ID NO:39變異的金黃色葡萄球菌殺白細胞素B(LukB)蛋白或多肽，所述LukB變異多肽包含: 對應於SEQ ID NO:39的氨基酸殘基Glu45、Glu109、Thr121和Arg154的一個或多個氨基酸殘基上的氨基酸取代。 A mutated Staphylococcus aureus leukocidin B (LukB) protein or polypeptide of SEQ ID NO: 39, the LukB mutated polypeptide comprising: Amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39.

如請求項49所述的LukB變異多肽，其中對應於Glu45的氨基酸殘基上的氨基酸取代包括穀氨酸到半胱氨酸(Glu45Cys)的取代，對應於Thr121的氨基酸殘基上的氨基酸取代包括蘇氨酸到半胱氨酸(Thr121Cys)的取代。The LukB variant polypeptide as described in claim 49, wherein the amino acid substitution corresponding to the amino acid residue of Glu45 comprises a substitution of glutamic acid to cysteine (Glu45Cys), and the amino acid substitution corresponding to the amino acid residue of Thr121 comprises Threonine to cysteine (Thr121Cys) substitution.

如請求項49或50所述的LukB變異多肽，其中對應於Glu109的氨基酸殘基上的氨基酸取代包括穀氨酸到半胱氨酸(Glu109Cys)的取代，對應於Arg154的氨基酸殘基上的氨基酸取代包括精氨酸到半胱氨酸(Arg154Cys)的取代。The LukB variant polypeptide as described in claim 49 or 50, wherein the amino acid substitution on the amino acid residue corresponding to Glu109 comprises a substitution of glutamic acid to cysteine (Glu109Cys), corresponding to the amino acid on the amino acid residue of Arg154 Substitutions include arginine to cysteine (Arg154Cys).

如請求項49~51中任一項所述的LukB變異多肽，其中所述LukB變異多肽包含對應於SEQ ID NO:39的氨基酸Glu45、Glu109、Thr121和Arg154的每個氨基酸殘基上的氨基酸取代。The LukB variant polypeptide according to any one of claims 49 to 51, wherein the LukB variant polypeptide comprises amino acid substitutions corresponding to each amino acid residue of Glu45, Glu109, Thr121 and Arg154 of SEQ ID NO:39 .

如請求項52所述的LukB變異多肽，其中所述變體是SEQ ID NO:15的CC8 LukB變體，其包含對應於SEQ ID NO:15的Glu45Cys、Glu109Cys、Thr121Cys和Arg154Cys的氨基酸取代。The LukB variant polypeptide according to claim 52, wherein the variant is the CC8 LukB variant of SEQ ID NO:15, which comprises amino acid substitutions corresponding to Glu45Cys, Glu109Cys, Thr121Cys and Arg154Cys of SEQ ID NO:15.

如請求項53所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:21的氨基酸序列。The LukB variant polypeptide as described in claim 53, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:21.

如請求項52所述的LukB變異多肽，其中所述變體是SEQ ID NO:16的CC45 LukB變體，包含對應於SEQ ID NO:16的Glu45Cys、Glu110Cys、Thr123Cys和Arg155Cys的氨基酸取代。The LukB variant polypeptide according to claim 52, wherein the variant is the CC45 LukB variant of SEQ ID NO:16, comprising amino acid substitutions corresponding to Glu45Cys, Glu110Cys, Thr123Cys and Arg155Cys of SEQ ID NO:16.

如請求項55所述的LukB變異多肽，其中所述LukB變異多肽包含SEQ ID NO:22的氨基酸序列。The LukB variant polypeptide as described in claim 55, wherein the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:22.

如請求項33~56中任一項所述的LukB變異多肽，還包含:氨基末端信號序列。The LukB variant polypeptide according to any one of claims 33 to 56, further comprising: an amino-terminal signal sequence.

如請求項57所述的LukB變異多肽，其中氨基末端信號序列包含SEQ ID NO:23的氨基酸序列。The LukB variant polypeptide as described in claim 57, wherein the amino-terminal signal sequence comprises the amino acid sequence of SEQ ID NO:23.

如請求項33~58中任一項所述的LukB變異多肽，還包含:氨基末端純化標簽。The LukB variant polypeptide according to any one of claims 33-58, further comprising: an amino-terminal purification tag.

一種編碼請求項33~59中任一項所述的LukB變異多肽的核酸分子。A nucleic acid molecule encoding the LukB variant polypeptide described in any one of claims 33-59.

一種包含請求項60所述的核酸分子的表達載體。An expression vector comprising the nucleic acid molecule described in claim 60.

一種表達載體，包括: 可操作地偶聯的請求項30所述的核酸分子與請求項60所述的核酸分子。 An expression vector comprising: The nucleic acid molecule of claim 30 and the nucleic acid molecule of claim 60 operably coupled.

一種包含請求項61或62所述的表達載體的宿主細胞。A host cell comprising the expression vector described in Claim 61 or 62.

一種金黃色葡萄球菌疫苗組合物，包含: 請求項1~29中任一項所述的一種或多種變異LukA蛋白或多肽。 A staphylococcus aureus vaccine composition comprising: One or more variant LukA proteins or polypeptides described in any one of claims 1-29.

如請求項64所述的疫苗組合物，其中所述LukA變異多肽是SEQ ID NO:1的變體。The vaccine composition as claimed in claim 64, wherein the LukA variant polypeptide is a variant of SEQ ID NO:1.

如請求項64或65所述的疫苗組合物，進一步包括: 一種殺白細胞素B(LukB)蛋白或多肽，所述LukB蛋白或多肽與SEQ ID NO:15的氨基酸序列具有至少85%的序列相似性。 The vaccine composition as described in claim 64 or 65, further comprising: A leukocidin B (LukB) protein or polypeptide having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO:15.

如請求項64或65所述的疫苗組合物，進一步包括: 一種殺白細胞素B(LukB)蛋白或多肽，所述LukB蛋白或多肽與SEQ ID NO:16的氨基酸序列具有至少85%的序列相似性。 The vaccine composition as described in claim 64 or 65, further comprising: A leukocidin B (LukB) protein or polypeptide having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO:16.

如請求項67所述的疫苗組合物，其中所述LukA變異多肽包含對應於SEQ ID NO:1的氨基酸殘基Lys80、Ser138、Val110、Val190和Glu320Ala的一個或多個氨基酸殘基上的氨基酸取代。The vaccine composition as claimed in claim 67, wherein the LukA variant polypeptide comprises amino acid substitutions corresponding to one or more amino acid residues of Lys80, Ser138, Val110, Val190 and Glu320Ala of amino acid residues of SEQ ID NO:1 .

如請求項68所述的疫苗組合物，其中所述LukA變異多肽包含SEQ ID NO:3的氨基酸序列，並且所述LukB多肽包含SEQ ID NO:18的氨基酸序列。The vaccine composition according to claim 68, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, and the LukB polypeptide comprises the amino acid sequence of SEQ ID NO:18.

如請求項68所述的疫苗組合物，其中所述LukA變異多肽包含SEQ ID NO:3的氨基酸序列，並且所述LukB蛋白或多肽包含SEQ ID NO:15的氨基酸序列。The vaccine composition according to claim 68, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:15.

如請求項68所述的疫苗組合物，其中所述LukA變異多肽包含SEQ ID NO:3的氨基酸序列，並且所述LukB蛋白或多肽包含SEQ ID NO:17的氨基酸序列。The vaccine composition according to claim 68, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:17.

如請求項64所述的疫苗組合物，其中所述LukA變異多肽是SEQ ID NO:2的變體。The vaccine composition as claimed in claim 64, wherein the LukA variant polypeptide is a variant of SEQ ID NO:2.

如請求項72所述的疫苗組合物，進一步包括: 一種殺白細胞素B(LukB)蛋白或多肽，所述LukB蛋白或多肽與SEQ ID NO:16的氨基酸序列具有至少85%的序列相似性。 The vaccine composition as described in claim item 72, further comprising: A leukocidin B (LukB) protein or polypeptide having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO:16.

如請求項72所述的疫苗組合物，進一步包括: 一種殺白細胞素B(LukB)蛋白或多肽，所述LukB蛋白或多肽與SEQ ID NO:15的氨基酸序列具有至少85%的序列相似性。 The vaccine composition as described in claim item 72, further comprising: A leukocidin B (LukB) protein or polypeptide having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO:15.

如請求項73所述的疫苗組合物，其中所述LukA變異多肽包含對應於SEQ ID NO:2的氨基酸殘基Lys81、Ser139、Val111、Val191和Glu321Ala的一個或多個氨基酸殘基上的氨基酸取代。The vaccine composition as claimed in item 73, wherein the LukA variant polypeptide comprises amino acid substitutions corresponding to one or more amino acid residues of Lys81, Ser139, Val111, Val191 and Glu321Ala of amino acid residues of SEQ ID NO:2 .

如請求項75所述的疫苗組合物，其中所述LukA變異多肽包含SEQ ID NO:4的氨基酸序列，並且所述LukB蛋白或多肽包含SEQ ID NO:16的氨基酸序列。The vaccine composition according to claim 75, wherein the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:4, and the LukB protein or polypeptide comprises the amino acid sequence of SEQ ID NO:16.

一種金黃色葡萄球菌疫苗組合物，包含: 請求項33~56中任一項所述的一種或多種變異LukB蛋白或多肽。 A staphylococcus aureus vaccine composition comprising: One or more variant LukB proteins or polypeptides described in any one of claims 33-56.

如請求項77所述的疫苗組合物，進一步包括: 一種殺白細胞素A (LukA)蛋白或多肽，所述LukA蛋白或多肽與SEQ ID NO:1 (CC8)的氨基酸序列具有至少85%的序列相似性。 The vaccine composition as described in claim item 77, further comprising: A leukocidin A (LukA) protein or polypeptide having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 1 (CC8).

如請求項77所述的疫苗組合物，進一步包括: 一種殺白細胞素A (LukA)蛋白或多肽，所述LukA蛋白或多肽與SEQ ID NO:2 (CC45)的氨基酸序列具有至少85%的序列相似性。 The vaccine composition as described in claim item 77, further comprising: A leukocidin A (LukA) protein or polypeptide having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 2 (CC45).

一種金黃色葡萄球菌疫苗組合物，包含: 請求項1~32中任一項所述的LukA變異多肽，和請求項33~56中任一項所述的LukB變異多肽。 A staphylococcus aureus vaccine composition comprising: The LukA variant polypeptide described in any one of claim items 1 to 32, and The LukB variant polypeptide described in any one of claims 33-56.

如請求項64~80中任一項所述的疫苗組合物，進一步包括: 佐劑。 The vaccine composition as described in any one of claim 64～80, further comprising: adjuvant.

如請求項64~81中任一項所述的疫苗組合物，進一步包括: 一種或多種額外的金黃色葡萄球菌抗原。 The vaccine composition as described in any one of claim 64～81, further comprising: One or more additional S. aureus antigens.

一種在受試者中產生針對金黃色葡萄球菌的免疫應答的方法，所述方法包括: 在所述受試者中有效產生針對金黃色葡萄球菌的所述免疫應答的條件下，向所述受試者施用請求項64~81中任一項所述的疫苗組合物。 A method of generating an immune response against Staphylococcus aureus in a subject, the method comprising: Under the condition that the immune response against Staphylococcus aureus is effectively generated in the subject, the vaccine composition of any one of claims 64-81 is administered to the subject.

如請求項64~81中任一項所述的疫苗組合物，其中所述疫苗組合物用於在受試者中產生針對金黃色葡萄球菌的免疫應答的方法中。The vaccine composition according to any one of claims 64-81, wherein the vaccine composition is used in a method for generating an immune response against Staphylococcus aureus in a subject.